Pretreatment method for detecting volatile organic compounds in biological sample

Abstract

The present invention relates to a pretreatment method for detecting volatile organic compounds in a biological sample. When a biological sample is pretreated using the method according to one aspect and volatile organic compounds in the pretreated sample are analyzed, it is possible to achieve sensitivity increase and uniformity of a matrix effect by only a simple step without the need to perform a complicated pretreatment step that is conventionally performed to increase analysis sensitivity. That is, the concentration of volatile organic compounds in a biological sample can be accurately measured with greater simplicity, thus reducing time, effort, and cost, and being very economical. In addition, the pretreatment method according to one aspect involves a simple process and enables accurate detection of the compounds thereafter, and thus can be used for monitoring research on in vivo exposure to volatile organic compounds.

Description

Pretreatment method for detecting volatile organic compounds in biological samples

[0001] This relates to a pretreatment method for detecting volatile organic compounds in biological samples.

[0002]

[0003] Volatile organic compounds (VOCs) are substances harmful to both ecosystems and the human body, causing numerous chronic and acute side effects such as central nervous system disorders, respiratory disorders, and leukemia, and in severe cases, can even induce cancer. They are primarily generated in the petrochemical, steel, and food industries. In particular, VOCs generate photochemical oxidants—secondary pollutants such as ozone—through photochemical reactions, which have harmful effects on the bodies of humans and animals. Since VOCs enter the human body and are primarily excreted through urine after undergoing metabolic processes, measuring the concentration of VOC metabolites in urine samples can indicate that an increase in concentration is an indication of VOC ingestion.

[0004] Solid Phase Micro-Extraction (SPME) and Purge & Trap methods are widely used for analyzing VOCs; these methods primarily employ a technique of concentrating the sample during the sample injection stage to increase instrument sensitivity. However, such analytical methods entail the inconvenience and burden of having to purchase additional equipment to enhance sensitivity.

[0005] Therefore, the inventors applied a simple, rapid, and highly reproducible method at the preprocessing stage that increases sensitivity, and also provides the effect of enhancing analysis precision by minimizing matrix effects that are difficult to achieve through equipment changes alone.

[0006] Generally, when it is difficult to analyze VOCs in aqueous solutions, salts such as NaCl are added during analysis. This method reduces the solubility of VOCs in the aqueous solution, thereby increasing sensitivity during instrumental analysis. However, even when analyzing serum and plasma rather than whole blood, there have been no cases where a method has been applied to induce protein denaturation in the sample by adding urea, thereby facilitating the release of VOCs bound to the proteins. A pretreatment method according to one aspect involved the application of urea, a reagent that induces protein denaturation, and salts of NaCl, Na2SO4, and K2SO4, which increase the release intensity of VOCs in the sample. It was ultimately developed with the aim of enhancing sensitivity and the uniformity of matrix effects.

[0007]

[0008] One aspect provides a method for pre-treating a sample for detecting volatile organic compounds in a biological sample, comprising the step of mixing one or more selected from the group consisting of a protein denaturation-inducing reagent and a salt into the isolated biological sample.

[0009] Another aspect provides a method for detecting volatile organic compounds in a separated biological sample, comprising the step of mixing one or more selected from the group consisting of a protein denaturation-inducing reagent and a salt into the separated biological sample.

[0010]

[0011] One aspect provides a method for pre-treating a sample for detecting volatile organic compounds in a biological sample, comprising the step of mixing a protein denaturation-inducing reagent and a salt into the isolated biological sample.

[0012] In one embodiment, the biological sample is separated from an individual, and the individual refers to a subject excluding all humans for whom volatile organic compound detection is required.

[0013] In one embodiment, the biological sample is whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate It may be one or more selected from the group consisting of aspirate, organ secretions, cell, cell extract, cerebrospinal fluid, and fur. Specifically, the biological sample may be one or more selected from the group consisting of whole blood, plasma, serum, urine, semen, and saliva, and more specifically, the biological sample may be whole blood.

[0014] The term "protein denaturation-inducing reagent" may also be referred to as denaturants and serves to destroy the tertiary structure of a protein to create a random coil state. This denatures the structure of the protein by destroying hydrogen bonds, hydrophobic interactions, and ionic bonds within the protein. A method according to one aspect involves mixing the protein denaturation-inducing reagent into the biological sample to induce protein denaturation and facilitate the release of volatile organic compounds bound to the protein.

[0015] In one embodiment, the protein denaturation-inducing reagent may be one or more selected from the group consisting of urea, SDS (Sodium dodecyl sulfate), guanidine hydrochloride (Guanidine-HCl), ethanol, acetone, isopropyl alcohol, and β-mercaptoethanol. Specifically, the protein denaturation-inducing reagent may be one or more selected from the group consisting of urea, SDS, and water, and more specifically, the protein denaturation-inducing reagent may be urea.

[0016] The above salt is mixed to induce a "salt-out effect," which refers to a phenomenon in which the solubility of a specific substance is reduced when a salt is added to a solution. A method according to one aspect adds a salt to the biological sample and induces a salt-out effect to reduce the solubility of volatile organic compounds in the sample, thereby promoting their movement from the aqueous phase to the gaseous phase. Accordingly, volatile organic compounds move more easily into the gaseous phase, increasing extraction efficiency.

[0017] In one embodiment, the salt is NaCl, Na2SO 4, K2SO 4,It may be one or more selected from the group consisting of Na2CO3, CaCl2, (NH4)2SO4, NH4Cl, and NaOAc. Specifically, the salt is NaCl, Na2SO4, and It may be one or more selected from the group consisting of K2SO4, and more specifically, the salt may be NaCl.

[0018] When performing a pretreatment method based on a specific pattern, the action of protein denaturation-inducing reagents and salts in the sample can increase analytical sensitivity during the analysis of volatile organic compounds and lead to uniformity of matrix effects.

[0019] The aforementioned "matrix effect" refers to the phenomenon in which complex components within a sample influence the detection and quantification of the target substance during the analysis of volatile organic compounds. Detection sensitivity may vary depending on the matrix components, and interference from other substances with properties similar to the target compound can occur. Furthermore, it can affect the extraction efficiency of the compound, which may result in variations in the recovery rate.

[0020] In one experimental example, for a sample mixed with urea and SDS among the protein denaturation-inducing reagents and NaCl, K2SO4, and Na2SO4 among the salts, the analytical sensitivity increased compared to a sample without such mixture, and the sensitivity variability with increasing sample volume was also low. In addition, the matrix effect within the sample was reduced and became more uniform (see Experimental Examples 1 to 3).

[0021] The aforementioned "Volatile Organic Compounds (VOCs)" are substances highly harmful to the human body as well as to the ecosystem, causing many chronic and acute side effects such as central nervous system disorders, respiratory disorders, and leukemia, and in severe cases, inducing cancer. They are mainly generated in the petrochemical, steel, and food industries, and in particular, VOCs have a significant impact on the human body by generating photochemical oxides, which are secondary pollutants such as ozone, through photochemical reactions.

[0022] In one embodiment, the volatile organic compound may be one or more selected from the group consisting of benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, styrene, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, and nonadecane.

[0023] In one embodiment, the detection may be performed using one or more methods selected from the group consisting of thermal desorption-gas chromatograph-mass spectrometry (TD-GC / MS), solid phase extraction (SPE), liquid-liquid extraction (LLE), solid phase micro-extraction (SPME), gas chromatograph-mass spectrometry (GC-MS or GC-MS / MS), purge and trap methods, and headspace-GC / MS. Specifically, the detection may be performed using headspace-GC / MS.

[0024] The aforementioned gas chromatography-mass spectrometry can simply and rapidly separate volatile substances from complex matrices, saving both time and cost. In addition, it has the advantage of being able to analyze volatile organic compounds in various forms of samples, such as liquids and solids.

[0025] In one embodiment, when performing the headspace gas chromatography mass spectrometry, a SIM (Selected Ion Monitoring) mode may be used for quantitative analysis, and when using GC-MS / MS, MRM (Multiple Reaction Monitoring) and SRM (Selected Reaction Monitoring) modes may be used by applying the ion transition determined from the mass spectrum.

[0026] In one embodiment, the pretreatment method may further include a step of mixing the biological sample and the anticoagulant prior to the mixing step.

[0027] In one embodiment, the anticoagulant serves to prevent the biological sample, particularly blood, from coagulating, and may be one or more selected from the group consisting of EDTA, citric acid, sodium heparin, lithium heparin, potassium oxalate, and sodium oxalate. Specifically, the anticoagulant may be one or more selected from the group consisting of EDTA and citric acid.

[0028] In one embodiment, the method may further include a step of refrigerating or freezing the separated biological sample prior to the mixing step.

[0029] In one embodiment, the detection refers not only to simply determining whether the target volatile organic compound is present in the separated biological sample—that is, identifying the volatile organic compound—but also to a quantitative measurement process that confirms the amount of the compound through ionization, mass analysis, and signal generation processes using a mass spectrometer.

[0030] Another aspect provides a method for detecting volatile organic compounds in a separated biological sample, comprising the step of mixing one or more selected from the group consisting of a protein denaturation-inducing reagent and a salt into the separated biological sample.

[0031] The above terms "biological sample," "protein denaturation-inducing reagent," "salt," "volatile organic compound," and "detection" may be within the aforementioned range.

[0032] In one embodiment, the detection may be performed using one or more methods selected from the group consisting of thermal desorption gas chromatographic mass spectrometry (TD-GC / MS), solid phase extraction (SPE), liquid-liquid extraction (LLE), solid phase trace extraction (SPME), gas chromatographic mass spectrometry (GC-MS or GC-MS / MS), purge and trap methods, and headspace-based gas chromatographic mass spectrometry (Headspace-GC / MS). Specifically, the detection may be performed using headspace-based gas chromatographic mass spectrometry (Headspace-GC / MS).

[0033]

[0034] When pre-treating biological samples and analyzing volatile organic compounds within the pre-treated samples using a method based on a single aspect, it is possible to ensure increased sensitivity and uniformity of matrix effects with only simple steps, without the need for complex pre-treatment steps previously performed to increase analytical sensitivity. In other words, the concentration of volatile organic compounds in biological samples can be accurately measured more simply, thereby reducing time, effort, and cost, and making it very economical. Furthermore, because the pre-treatment method based on a single aspect is simple and the subsequent detection of compounds is accurate, it can be utilized in studies such as monitoring biological exposure to volatile organic compounds.

[0035]

[0036] Figure 1 shows the matrix effect of fluorobenzene in 63 samples without the addition of urea and each salt.

[0037] Figure 2 shows the results of comparing the matrix effects of each combination of fluorobenzene in blood samples to which urea and each salt were added.

[0038] Figure 3 shows the matrix effect of fluorobenzene in 40 samples with added urea and NaCl.

[0039] Figures 4 to 22 show the GC-MS SIM Chromatogram results of volatile organic compounds detected in blood samples to which urea, SDS, and each salt substance were added.

[0040]

[0041] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0042]

[0043] Reference Example

[0044] Reference Example 1. Performing blood sample storage and pretreatment steps

[0045] VOCs in blood samples were measured, and the sample storage and pretreatment process prior to measurement is as follows.

[0046] Anticoagulants such as citric acid and EDTA (ethylene-diamine-tetraacetic acid) were used to prevent blood samples from coagulating, and the samples were refrigerated at 4°C or below immediately after collection until analysis.

[0047] Next, urea, SDS, and water—reagents that induce protein denaturation—and NaCl, Na2SO4, K2SO4, and water—salts that increase the emission intensity of VOCs in the sample—were added in combination to a 20 mL headspace analysis vial containing a blood sample. This was done to increase analytical sensitivity by reducing the solubility of volatile organic compounds in the aqueous analysis sample through the salt-out effect caused by the addition of salts. The specific combinations of added reagents are shown in Table 1 below.

[0048]

[0049] Combination Protein Denaturation Inducing Reagent Salts Example 1 Urea NaCl Example 2 Urea K2SO4 Example 3 Urea Na2SO4 Example 4 SDSNaCl Example 5 SDSK2SO4 Example 6 SDSNa2SO4 Comparative Example 1 H2ONaCl Comparative Example 2 H2OK2SO4 Comparative Example 3 H2ONa2SO4 Comparative Example 4 Urea H2O Comparative Example 5 SDSH2O Comparative Example 6 H2OH2O

[0050] Reference Example 2. Setting Specific Conditions for GC-MS Analysis

[0051] Gas chromatography-mass spectrometry (GC-MS) was used to analyze volatile organic compounds (VOCs) in blood.

[0052] For quantitative analysis using GC-MS, SIM (Selected Ion Monitoring) mode was used, and the specific Headspace-GC-MS analysis conditions are as shown in Table 2 below.

[0053]

[0054] GCThermoscientific TRACE 1610MSDThermoscientific ISQ 7610HeadspaceThermoscientific TriPlus 500ColumnVF-624ms capillary column (0.32 mm id x 30 m, 0.18 μm film thickness)Oven(Programming)Rate 1: from 50 ℃(5 min) to 100 ℃ at 10 ℃ / minRate 2: from 100 ℃ to 120 ℃ at 20 ℃ / minRate 3: from 120 ℃ to 260 ℃(4.33 min) at 30 ℃ / minInlet Temp.(℃)250 ℃Septum Purge Flow2 mL / minMS Transfer Line Temp.200 ℃Ion Source Temp.260 ℃Carrier gasHelium, 2 mL / minInjection modeSplit mode, Split ratio 20

[0055]

[0056] Reference Example 3. Selection of Quantitative and Qualitative Ions in GC-MS Analysis

[0057] A total of 19 components were used for the measurement of VOCs in blood: benzene, toluene, ethylbenzene, m-, p-xylene (m-xylene and p-xylene), o-xylene, styrene, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, and nonadecane. Fluorobenzene, which is not present in blood but possesses physicochemical properties similar to the analytes, was used as the internal standard. However, the internal standard for the quantification of VOCs in blood was not limited to fluorobenzene, and for each component 13Isotope-labeled standards with isotopes such as C and deuterium can be used, and the Rt. (Retention time) and quantitative and qualitative ions of each component analyzed by applying the instrumental analysis conditions of Table 1 above are as shown in Table 3 below.

[0058]

[0059] CompoundMolecularweightRt.Quantitativeion(m / z)Qualitativeion(m / z)Benzene78.114.787877Toluene92.148.089192Ethylbenzene106.1710.489 1106m-,p-xylene106.1610.68106105o-xylene106.1611.1691106Styrene104.1511.2410478Octane114.238.194385Nonane128.210.675743Decane142. 2912.165743Undecane156.3113.125743Dodecane170.3413.845743Tridecane184.414.445771Tetradecane198.3914.975771Pentadecane212.4215.455 771Hexadecane226.4515.897185Heptadecane240.4716.315771Octadecane254.4916.725771Nonadecane268.5217.175771Fluorobenzene96.15.479670

[0060]

[0061] Experimental Example 1. Comparison of Sensitivity by Combination for GC-MS Results

[0062] The sensitivity to each volatile organic compound of Examples 1 to 6 and Comparative Examples 1 to 6 was confirmed.

[0063] 12.8 mL of a saturated solution of NaCl, Na2SO4, and K2SO4 containing 2.61 M urea was added to a 20 mL vial for headspace analysis containing 1.5 g of blood sample, and this was used as the sample for Headspace-GC-MS analysis. The sample was heated to 99 °C for 40 minutes in an incubator within the instrument, after which 1 mL of the top gas from the vial was injected into the GC-MS. To separate VOCs loaded onto the capillary column, the column oven temperature was held at 50 °C for 5 minutes, then increased to 100 °C at a rate of 10 °C per minute, and further increased to 120 °C at a rate of 20 °C per minute. Subsequently, the temperature was increased from 120 °C to 260 °C at a rate of 30 °C per minute, followed by a holding period of 4.33 minutes, applying a final heating condition of 20 minutes.

[0064] First, the analytical sensitivity in Headspace-GC-MS in Examples 1 to 6 was compared. It was evaluated as the ratio of the reactions of Examples 1 to 6 and Comparative Examples 1 to 5 to the reaction of the sample dissolved in water, i.e., the sensitivity of Comparative Example 6, and was calculated by the following Equation 1:

[0065] [Mathematical Formula 1]

[0066] Recovery(%) = Example or Comparative Example / Comparative Example 6 * 100

[0067]

[0068] The results of confirming sensitivity for each volatile organic compound are shown in Tables 4 and 5 below. The sensitivity confirmation experiment was repeated a total of three times, and cases with large p-values ​​were excluded from Tables 4 and 5 below as they could not be interpreted as significant data.

[0069]

[0070] Compound Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Benzene 153.29 2.0 103.0 -120-Fluorobenzene 141.2 104.3 122.4 ---Toluene 75.0 -----Octane 246.4 201.3 126.7 -170-Ethylbenzene 159.0 110.8 --110-m-,p-xylene 165.7 112.3 --110-Nonane 296.4 242.9 144.8 -160-o-xylene 175.9 111.4 101.8 -110-Styrene 179.0 -102.0 ---Decane 303.6 257.3158 .7-190-Undecane339.4265.8178.4-170-Dodecane303.5240.8180.1-300100Tridecane281.7220.7195.5-210-Tetradecane242.8182.4174.24201400540Pentadecan e139.2122.9115.0100490140Hexadecane91.9102.7116.34501190550Heptadecane 106.2122.3109.7130850160Octadecane46.5--170950170Nonadecane50.9---890-

[0071]

[0072] Compound Comparison Example 1 Comparison Example 2 Comparison Example 3 Comparison Example 4 Comparison Example 5 Comparison Example 6 Benzene---70 90 100 Fluorobenzene-----100 Toluene 90 100 90 60 90 100 Octane 120 90 40--100 Ethylbenzene 90 70 70 90 100 m-,p-xylene 90 60 70 90 100 Nonane--40 100 o-xylene 100 70 70 80 100 Styrene e--1007070100Decane--50--100Undecane--50--100Dodecane--50--100Tridecane--60--100Tetradecane--90--10 0Pentadecane---90-100Hexadecane---70-100Heptadecane---60-100Octadecane---40-100Nonadecane--10010-100

[0073]

[0074] In the case of Example 1, except for toluene, octadecane, nonadecane, and hexadecane, which were evaluated to have low sensitivity compared to the sample dissolved in water among the VOC components, an overall high sensitivity increase effect of 139.2% to 339.4% was observed.

[0075] In Example 2, undecane showed the highest sensitivity increase effect of 265.8%, and in Example 3, tridecane showed the highest sensitivity increase effect of 195.5%.

[0076] In Example 4, a high sensitivity increase effect was observed particularly in Tetradecane and Hexadecane; in Example 5, a high sensitivity increase effect was observed particularly in Tetradecane, Hexadecane, Heptadecane, Octadecane, and Nonadecane; and in Example 6, an excellent sensitivity increase effect was observed in Tetradecane and Hexadecane. That is, in all of Examples 1 to 6, sensitivity increased in most components compared to the sample dissolved in water and the sample containing the protein denaturation-inducing reagent or salt alone.

[0077]

[0078] Experimental Example 2. Analysis of Sensitivity Change According to Sample Amount

[0079] Next, the degree to which the sensitivity to each volatile organic compound changed as the amount of samples of Examples 1 to 6 and Comparative Examples 1 to 6 increased was confirmed.

[0080] Blood samples of 0.5 g and 1.5 g were used. After adding the same concentration of STD to each sample, the percentage change in sensitivity measured in the 1.5 g sample was measured relative to the sensitivity measured in the 0.5 g sample. The results were calculated using Equation 2 below and are shown in Tables 6 and 7 below. The measurement of the change in sensitivity was repeated a total of three times. Samples with large p-values ​​were excluded from Tables 4 and 5 below as they could not be interpreted as significant data.

[0081]

[0082] [Mathematical Formula 2]

[0083] Degree of change in sensitivity (%) = (Sensitivity measured in 1.5 g blood sample - Sensitivity measured in 0.5 g blood sample) / Sensitivity measured in 0.5 g blood sample * 100.

[0084]

[0085] Compound Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Benzene 3 1.4 9.4 19.2 14.7 Toluene 3 4.8 50.9 44.0 --- Octane 2 0.5 33.2 - 10.9 25.9 10.8 Ethylbenzene 7.7 9.7 10.9 24.3 - 16.9 m-,p-xylene 8.9 8.9 9.4 23.9 - 15.1 Nonane 19.3 36.1 - 20.8 25.3 4.1 o-xylene 9.6 7.1 2.2 ---Styrene 2 0.0 9.2 8.0 - 15.5 Decane 17.4 -- 17.0 0.4 14.4 Undecane 20.433.0-10.437.6-Dodecane33.027.1-27.18.521.9Tridecane61.217.6-30.4-39.1Tetradecane39.623.1-38.72.66.1Pentadecane6.014.7-11.9--Hexa decane24.413.3-28.3-1.7Heptadecane16.02.9-5.5-5.4Octadecane59.217.4-0.1-1.8Nonadecane44.932.237.246.1-37.1Fluorobenzene0.46.41.8-6.0-

[0086]

[0087] Compound Comparison Example 1 Comparison Example 2 Comparison Example 3 Comparison Example 4 Comparison Example 5 Comparison Example 6 Benzene 3 1.4 9.4 19.2 - 45.8 - Toluene 5 0.2 6 2.7 - 107.6 11.5 Octane 3 3.2 5 1.2 - 25.9 - Ethylbenzene 3 3.3 - 58.5 48.0 - 29.1 - xylene 3 5.9 - 60.2 48.9 - 29.4 Nonane 5 8.1 62.9 63.4 - 46.2 - xylene 3 6.2 - 59.74 7.6--Styrene31.0-57.342.731.129.3Decane23.356.839.555.2-52.0Undecane-57.0-49.6-54.0Dodecane-60.0-41.7- 59.3Tridecane-63.6---67.7Tetradecane-67.3--42.977.4Pentadecane22.068.428.332.5-80.7Hexadecane-69.639.8- 42.481.8Heptadecane-69.5- 31.8-82.6Octadecane-74.9- - - 86.4Nonadecane-77.3- 60.4- 88.4Fluorobenzene28.911.217.930.3-21.4

[0088]

[0089] As a result of the verification, it was confirmed that the degree of sensitivity variability according to the amount of blood sample was generally lower in Examples 1 to 6 compared to Comparative Examples 1 to 6, and among all components, particularly regarding Benzene, Toluene, Ethylbenzene, Xylene, and Styrene, it was confirmed that the variability as the amount of blood sample increased was smallest in Examples 1 to 3.

[0090]

[0091] Experimental Example 3. Comparison of the uniformity of matrix effects for each combination based on GC-MS results

[0092] Among Examples 1 to 6, in which the increase in sensitivity and the degree of variation according to volatile organic compounds were confirmed in Experimental Examples 1 and 2 above, the degree of uniformity of the matrix effect according to the combination of the protein denaturation-inducing reagent and salt of Examples 1 to 3 was confirmed.

[0093] We attempted to experimentally create various matrices by varying the amount of blood samples, and confirmed the level of matrix effect when each combination of Examples 1 to 3 in Table 1 was applied and analyzed on the various matrices. For the evaluation of the matrix effect, fluorobenzene, an internal standard that is not present in blood but has physicochemical properties similar to the components being analyzed, was used.

[0094] Assuming the use of a 1 mL blood sample, and considering cases where the matrix amount is less or more than this, the blood samples were quantified to 0.5 g and 1.5 g, respectively, for analysis. The matrix effect was evaluated by dividing the area value of samples with varying blood weights by the area value of samples with water applied instead of blood to assess the degree of sensitivity increase or decrease in blood samples. The specific matrix effect was calculated using the following Equation 3:

[0095] [Mathematical Formula 3]

[0096] Matrix Effect(%) = (Blood Sample - Non-blood Sample) / Non-blood Sample * 100

[0097]

[0098] First, when 1 mL of blood without urea was applied to 63 actual blood samples for analysis, it was confirmed that the matrix effect of fluorobenzene was distributed very differently from 44% to -81.6% (Fig. 1).

[0099] Next, the matrix effect was confirmed at 0.5 g and 1.5 g of blood samples in Examples 1 to 3. As a result, in Example 1, which mixed urea and NaCl, the values ​​were 10.5% and 41.2%, respectively; in Example 2, which mixed urea and K2SO4, the values ​​were 22.9% and 4.3%, respectively; and in Example 3, which mixed urea and Na2SO4, the values ​​were 2.1% and 22.4%, respectively. In other words, all examples showed an improved matrix effect within a narrower range than when no reagent was applied (Fig. 2). Subsequently, the urea and NaCl from Example 1, which showed the highest sensitivity as confirmed in Experimental Example 1, were applied to 1 mL of an actual blood sample, and the level of variance of the matrix effect was evaluated by Headspace-GC-MS. As a result, when 40 samples were analyzed, a matrix effect distribution ranging from a maximum of 25% to -35.5% was observed, confirming that the matrix effect was reduced compared to samples without urea and NaCl applied in the actual sample application analysis (Fig. 3).

[0100] Consequently, it can be observed that the matrix effect appears within a narrower range, that is, uniformly, when elements are mixed, compared to when elements are not included.

[0101]

[0102] Experimental Example 4. Single Peak Analysis of Volatile Organic Compounds

[0103] In addition, the single peak results of each volatile organic compound component in Examples 1 to 6 were confirmed. As a result, it was confirmed that all volatile organic compounds exhibited excellent symmetry and selectivity in their single peaks (Figs. 4 to 22). That is, when chromatography is performed using the above method, interference between substances is minimized, allowing for clear separation of components and facilitating the identification of the target compound. Furthermore, the excellent selectivity indicates that the signal of the target compound is hardly distorted by other components, and that accurate qualitative analysis has been performed.

[0104] In other words, although there were differences in sensitivity depending on the combination, all combinations showed improved sensitivity and increased uniformity of the matrix effect, and it was confirmed that measurement at very low concentrations in the ng / mL range was possible.

Claims

1. A method for pre-treating a sample for detecting volatile organic compounds in a biological sample, comprising the step of mixing a protein denaturation-inducing reagent and a salt into a separated biological sample.

2. In Claim 1, the biological sample comprises whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint A method comprising one or more selected from the group consisting of joint aspirate, organ secretions, cell, cell extract, cerebrospinal fluid, and fur.

3. The method of claim 1, wherein the protein denaturation-inducing reagent is one or more selected from the group consisting of urea, SDS (Sodium dodecyl sulfate), guanidine hydrochloride (Guanidine-HCl), ethanol, acetone, isopropyl alcohol, and β-mercaptoethanol.

4. In Claim 3, the salt is NaCl, Na2SO 4, K2SO 4, A method comprising one or more selected from the group consisting of Na2CO3, CaCl2, (NH4)2SO4, NH4Cl and NaOAc.

5. The method of claim 1, wherein the volatile organic compound is one or more selected from the group consisting of benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, styrene, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, and nonadecane.

6. The method of claim 1, wherein the detection is performed using one or more selected from the group consisting of thermal desorption-gas chromatograph-mass spectrometry (TD-GC / MS), solid phase extraction (SPE), liquid-liquid extraction (LLE), solid phase micro extraction (SPME), gas chromatograph-mass spectrometry (GC-MS or GC-MS / MS), purge and trap method, and headspace-GC / MS.

7. The method of claim 1, further comprising the step of mixing the biological sample and the anticoagulant prior to the mixing step.

8. The method of claim 7, wherein the anticoagulant is one or more selected from the group consisting of EDTA, citric acid, sodium heparin, lithium heparin, potassium oxalate, and sodium oxalate.

9. A step of obtaining a mixture by mixing a protein denaturation-inducing reagent and a salt with a separated biological sample; and A method for detecting volatile organic compounds in a biological sample, comprising the step of detecting the amount of volatile organic compounds present in the mixture.

10. The method of claim 9, further comprising the step of mixing the biological sample and the anticoagulant prior to the step of obtaining the mixture.

11. A method according to claim 9, wherein the detection is performed using one or more selected from the group consisting of thermal desorption-gas chromatograph-mass spectrometry (TD-GC / MS), solid phase extraction (SPE), liquid-liquid extraction (LLE), solid phase micro extraction (SPME), gas chromatograph-mass spectrometry (GC-MS or GC-MS / MS), purge and trap method, and headspace-GC / MS.

12. The method of claim 9, wherein the volatile organic compound is one or more selected from the group consisting of benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, styrene, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, and nonadecane.

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