KR102120822B1 - Analytical method for the simultaneous determination of trimethylamine N-oxide and its related compounds in dried blood spots - Google Patents

Abstract

The present invention relates to a simultaneous quantitative analysis method for a trimethylamine oxide-based compound in dried blood spots. The method includes: (a) a step of performing hydrochloric acid treatment on dried blood spot (DBS) filter paper; (b) a step of dripping blood on the DBS filter paper; (c) a step of obtaining a supernatant by adding an internal standard solution and an elution solvent to the blood-dripped DBS filter paper, causing a reaction therebetween, and then performing centrifugation; (d) a step of adding ethyl bromoacetate (EtBAc) to the obtained supernatant and causing a reaction therebetween; and (e) a step of analyzing the reactant obtained in step (d). Simultaneous quantitative analysis of endogenous compounds for cardiovascular diseases can be performed with the present invention, which is expected to be useful for diagnosis and analysis of various cardiovascular diseases based on excellent detection sensitivity, linearity, recovery rate, repeatability, and stability.

Description

{Analytical method for the simultaneous determination of trimethylamine N-oxide and its related compounds in dried blood spots}

The present invention relates to a method for simultaneous quantitative analysis of a cardiovascular disease compound in dry drop blood, and specifically to a method for simultaneous quantitative analysis of a trimethylamine oxide compound in dry drop blood.

Since a recent study on the relationship between trimethylamine N -oxide (TMAO) and atherosclerosis was published (2011), various compounds related to TMAO and TMAO metabolism have been involved in disease mechanisms. It is said that. In addition, various cardiovascular diseases such as myocardial infarction, stroke, thromobosis, heart failure, arrhythmia such as atrial fibrillation are closely related to these compounds. It is revealed newly.

Therefore, TMAO and TMAO metabolic compounds trimethylamine (TMA), carnitine (car), choline (CHL), betaine (betaine; BET), γ-butylrobetaine (γ-butyrobetaine) ; GBB), trimethyllysine (TML), homocysteine (HCY), dimethylglycine (DMG), etc., have been reported as potential biomarkers associated with cardiovascular disease. Interest in these compounds is increasing as research results have been published that not only enzymes in the human body but also gut microbiome are involved in metabolism of human body.

Accordingly, the demand for simultaneous analysis of various compounds related to TMAO and TMAO metabolism will also gradually increase, and there is a need for continuous research related to this.

Dry blood spots (DBS) are samples that are dried by dropping a small amount of blood on standardized filter paper, and the sampling process is more convenient and economical compared to conventional biological samples such as plasma or blood. It has the advantage of easy storage and transportation of samples.

With the rapid development of current analytical technology, the scope of clinical application of DBS is gradually expanding from newborn screening using DBS, monitoring of blood drug concentration (TDM) and diagnosis of various diseases. In particular, since continuous monitoring is required when diagnosing metabolic diseases such as cardiovascular diseases, DBS capable of directly collecting a sample may be more useful than a conventional sampling method.

However, DBS has low analytical sensitivity due to the small amount of sample, and it is difficult to secure a blank matrix as the target component is an endogenous substance, and trimethylamine (TMA), a TMAO-related compound, is LC-ESI (electrospray ionization) due to volatility. )-MS Ionization or loss in the process of DBS production was not easy to develop the test method.

Therefore, it is possible to simultaneously quantitatively analyze various compounds related to TMAO and TMAO metabolism, and there is a need for research on analytical methods excellent in detection sensitivity for volatile compounds.

1. Republic of Korea Registered Patent No. 10-1505064

The object of the present invention is trimethylamine N-oxide (trimethylamine N -oxide; TMAO) and TMAO metabolism can be analyzed simultaneously for a variety of compounds, and the detection sensitivity for volatile compounds also excellent cardiovascular compounds simultaneous quantitative analysis method To provide.

In order to achieve the above object, the present invention comprises the steps of: (a) drying blood spots (DBS) filter paper hydrochloric acid treatment; (b) dripping blood onto the DBS filter paper; (c) reacting by adding an internal standard solution and an elution solvent to the blood-dropped DBS filter paper, followed by centrifugation to obtain a supernatant; (d) reacting by adding ethyl bromoacetate (EtBAc) to the obtained supernatant; And (e) analyzing the reactants obtained in the step (d); provides a method for simultaneous quantitative analysis of trimethylamine oxide compounds in the blood.

The method of simultaneous quantitative analysis of trimethylamine oxide-based compounds in blood according to the present invention uses dry blood spots (DBS) as an analysis object, thereby sampling the sample compared to a conventional biological sample such as plasma or blood. This is convenient, economical, and easy to store or transport samples.

In addition, the simultaneous quantitative analysis method of trimethylamine oxide compounds in blood according to the present invention prevented volatilization of volatile substances by using hydrochloric acid before blood dripping, and also introduced chemical derivatization methods to improve the detection sensitivity of compounds.

In addition, the trimethylamine oxide-based compound is an endogenous compound for cardiovascular diseases, and 9 related compounds (trimethylamine N -oxide (TMAO), trimethylamine (TMA), carnitine (Car); Choline (CHL), Betaine (BET), γ-butyrobetaine (GBB), trimethyllysine (TML), homocysteine (HCY), dimethylglycine (DMG) )) can be used for simultaneous quantitative analysis, and it can be useful for diagnosis and analysis of various cardiovascular diseases because of its excellent linearity, recovery rate, repeatability, and stability.

1 is a view showing a comparison of the reaction of each of the six derivatization reagents at a concentration of 1.0 mol/L, which shows the best detection sensitivity in all nine components to be analyzed.
2 is a view showing the effect of recovery of trimethylamine according to the concentration of hydrochloric acid in a Whatman 903 protein saver card dry blood drop (DBS) filter paper.
FIG. 3 is an LC-HRMS chromatogram of a stable isotope labeled compound (surrogate analyte; SA) and an internal standard for endogenous substances; (a) HCY-d ₄ 0.3 μmol/L, (b) DMG-d ₆ 0.3 μmol/L, (c) BET-d ₉ 0.15 μmol/L, (d) GBB-d ₉ 0.06 μmol/L, (e ) CAR-d ₃ 0.3 μmol/L, (f) TMA-d ₉ 0.03 μmol/L, (g) CHL-d ₉ 0.075 μmol/L, (h) TML-d ₉ 0.3 μmol/L, (i) TMAO It is a diagram showing -d ₉ 0.6 μmol/L and (j) COT-d ₃ (IS).
4 is a diagram showing the linearity of (A) HCY-d ₄ , (B) DMG-d ₆ and (C) BET-d ₉ .
5 is a diagram showing the linearity of (A) GBB-d ₉ , (B) CAR-d ₃ and (C) TMA-d ₉ .
6 is a diagram showing the linearity of (A) CHL-d ₉ , (B) TML-d ₉ and (C) TMAO-d ₉ .
FIG. 7 is a diagram showing the results of the stability of a standard solution of (A) 9 kinds of real endogenous substances (authentic analyte; AA), and (B) 9 kinds of endogenous substances.
8 is a view showing the results of an autosampler stability of a stable isotope-labeled compound (surrogate analyte; SA) for 9 endogenous substances.
FIG. 9 is a diagram showing short-term stability results of a stable isotope-labeled compound (surrogate analyte; SA) for nine endogenous substances.
FIG. 10 is a diagram showing long-term stability results of a stable isotope-labeled compound (surrogate analyte; SA) for 9 endogenous substances.

Hereinafter, the present invention will be described in detail.

The present inventors have introduced a chemical derivatization method through prevention of volatilization through pretreatment of hydrochloric acid and treatment with ethyl bromoacetate (EtBAc) as a pretreatment of dry drop blood (DBS), to improve detection sensitivity. Using the Korean stable isotope-labeled compound (surrogate analyte; SA), it is possible to accurately and simultaneously quantitatively analyze even a small amount of endogenous substances, as well as excellent linearity, recovery, repeatability and stability, resulting in myocardial infarction, stroke , Thrombosis (thromobosis), heart failure (heart failure), atrial fibrillation (atrial fibrillation) such as arrhythmia (arrhythmia) has been found to be useful in the diagnosis and analysis of various cardiovascular diseases, and completed the present invention.

The present invention comprises the steps of (a) hydrochloric acid treatment of filtered blood spots (DBS) filter paper; (b) dripping blood onto the DBS filter paper; (c) reacting by adding an internal standard solution and an elution solvent to the blood-dropped DBS filter paper, followed by centrifugation to obtain a supernatant; (d) reacting by adding ethyl bromoacetate (EtBAc) to the obtained supernatant; And (e) analyzing the reactants obtained in step (d); It provides a method for simultaneous quantitative analysis of trimethylamine oxide compounds in the blood containing.

At this time, the trimethylamine oxide-based compound is trimethylamine N-oxide (trimethylamine N -oxide; TMAO), trimethylamine (trimethylamine; TMA), carnitine (carnitine; CAR), choline (choline; CHL), betaine (betaine; BET) ), γ-butyrobetaine (GBB), trimethyllysine (TML), homocysteine (HCY), dimethylglycine (DMG), but is not limited thereto.

In addition, the step of treating the dried blood spots (DBS) filter paper with hydrochloric acid may be treated with hydrochloric acid at a concentration of 0.1 to 1.0 mol/L, but preferably with a concentration of 0.5 mol/L. .

The hydrochloric acid treatment is intended to use an acid-base neutralization reaction, and TMA is a basic volatile component in itself, but when reacted with an acid that is hydrochloric acid, salts are generated and may exist in a solid phase even at room temperature, thereby preventing volatility.

In addition, the step of reacting by adding ethyl bromoacetate (EtBAc) may treat EtBAc at a concentration of 0.75 to 1.5 mol/L, but may preferably be treated at a concentration of 1.0 mol/L. .

The treatment of ethyl bromoacetate (EtBAc) can increase the detection sensitivity using a chemical derivatization reaction.

At this time, if the filter paper treatment and derivatization compound treatment conditions are exceeded, the volatile compounds in the dried blood spots (DBS) volatilize, resulting in a low recovery rate, or the derivatization reaction is not properly performed, resulting in poor detection sensitivity. In addition, the effect of the recovery rate and detection sensitivity compared to the used compound is not good, which may cause uneconomical problems.

In addition, the step of adding an internal standard solution and an elution solvent to the blood-dropped DBS filter paper may further include a dithiothreitol (DTT) reducing agent, preferably 5 mmol/L. It may be included as a concentration, but is not limited thereto.

In addition, the step of analyzing the reactant is characterized by analyzing by chromatography/mass spectrometry, and preferably, liquid chromatography-high resolution mass spectrometry (LC-HRMS) may be used. , But is not limited thereto.

In addition, the analysis method is characterized by improving the detection sensitivity by 2 to 86 times.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, according to the gist of the present invention, the scope of the present invention is not limited by these examples to those skilled in the art to which the present invention pertains. It will be obvious.

<Reference example> Reagents and equipment

Actual endogenous substance (authentic analyte; AA) of trimethylamine N - oxide (trimethylamine N -oxide; TMAO), trimethylamine hydrochloride (trimethylamine hydrochloride; TMA · HCl) , carnitine (carnitine; CAR), choline chloride (choline chloride; CHL Cl), betaine (BET), γ-butyrobetaine hydrochloride (GBBHCl), trimethyllysine hydrochloride (TMLHCl), homocysteine (HCY), dimethyl glycine (dimethylglycine; DMG) standard was Sigma-Aldrich Corporation (St. Louis, MO, USA) from the stabilizing labeled compound for an endogenous substance (surrogate analyte; SA) of trimethylamine N - oxide -d ₉ (trimethylamine N -oxide-d ₉ ; TMAO-d _9), trimethylamine hydrochloride -d _{9 (trimethylamine-d 9 hydrochloride; TMA} -d 9 · HCl), ₃ -d carnitine hydrochloride _{(carnitine-d 3 hydrochloride; CAR} -d 3 · HCl), choline -d ₉ chloride _{(choline-d 9 chloride; CHL} -d 9 · Cl), betaine -d ₉ chloride _{(betaine-d 9 chloride; BET} -d 9 · Cl), γ- butyl -d ₉ betaine hydrochloride (γ _{-butyrobetaine-d 9 chloride; GBB-} d 9 · Cl), trimethyl lysine _{-d 9 (trimethyllysine-d 9;} TML-d 9), homocysteine _{-d 4 (homocysteine-d 4;} HCY-d 4), dimethylglycine -d ₆ hydrochloride _{(dimethylglycine-d 6 hydrochloride; DMG} -d 6 · HCl) were obtained from each standard CDN Isotopes Inc. (Quebec, Canada). Cotinine -d ₃ was used as the internal standard (internal standard, IS) (cotinine -d 3; COT-d 3) standard was purchased from Sigma-Aldrich Company. Methyl bromoacetate (MeBAc), ethyl bromoacetate (EtBAc), isopropyl bromoacetate (iPrBAc), tert -butyl bromoacetate (tBuBAc), Phenyl bromoacetate (PhBAc), benzyl bromoacetate (BzBAc) and sodium carbonate, sodium bicarbonate, ammonium hydroxide solution, hydrochloric acid, PBS (phosphate-buffered saline) and dithiothreitol were purchased from Sigma-Aldrich, water, methanol, acetonitrile and formic acid, acetic acid , Ammonium acetate, ammonium formate was used by LC-MS class of Thermo Fisher Scientific (Fair Lawn, NJ, USA) All other reagents were purchased by express or analysis.

< Example 1> DBS pre-treatment method

40 μL of 0.5 mol/L hydrochloric acid solution is added to each hole of the DBS filter paper Whatman 903 protein saver card, dried at room temperature for 2 hours, and 30 μL of blood is added dropwise and again It was dried at room temperature for 2 hours. DBS discs made by punching dried filter paper with a 6 mm diameter puncher were placed in a 1.5 mL microtube. Internal standard solution (COT-d ₃ , 1 μg/mL) 10 μL and 20 mmol/L ammonium formate (pH 9.0) Elution Solvent 370 μL, 5 mmol/L dithiothreitol ; DTT) 20 μL of reducing agent was added for 10 minutes, agitation was performed, and the mixture was left at room temperature for 20 minutes, and then centrifuged at 13,000 rpm for 3 minutes, and 200 μL of supernatant was transferred to another 1.5 mL microtube. To this, 100 μL of a 1.0 mol/L ethyl bromoacetate (EtBAc) solution was added, stirred for 5 minutes, and derivatized in an 80°C water-bath for 1 hour, followed by microtube ( The microtube) was taken out of the water-bath. After the reaction was completed, 200 mmol of 30 mmol/L formic acid (dissolved in acetonitrile) was added and stirred for 5 minutes, followed by centrifugation at 13,000 rpm for 5 minutes to remove the supernatant from acetonitrile and 1: Mix at 4 ratios (supernatant/acetonitrile, v/v). It was filtered through a syringe filter (0.2 μm, PTFE) and injected into LC-HRMS.

< Example 2> LC- HRMS analysis

Established LC conditions that can smoothly separate 9 types of components to be analyzed with optimal sensitivity. Since the target components are all polar compounds, three types of HILIC fixed phase (zwitterion, bare silica) were compared. First, zwitterion type SeQuant ZIC-HILIC column (100 mm × 2.1 mm id, 3.5 μm particle size), Obelisc R column (150 mm × 2.1 mm id, 5 μm particle size) and silica (bare silica) ) Type of Acquity UPLC BEH HILIC column (100 mm × 2.1 mm id, 1.7 μm particle size) was selected, and the optimal mobile phase conditions were established and compared for each column. As a result, the Obelisc R column was excluded from the target stationary phase due to the peak broadness phenomenon of HCY, and the ZIC-HILIC column with better overall analysis sensitivity and peak sharpness among the ZIC-HILIC column and Acquity UPLC BEH HILIC column was finally selected as the target stationary phase. Did. As a mobile phase additive, ammonium formate and ammonium acetate were compared. As a result of testing by adjusting the concentration of both additives to 2 mmol/L and the pH to 3.0 respectively, ammonium formate showing a better peak shape was finally selected as a mobile phase additive. In addition, by adjusting the gradient elution conditions and column temperature, optimal LC conditions for smoothly separating 9 target components were established.

In this test, 9 types of target components were analyzed using Q-Exactive of Thermo Fisher Scientific by HRMS. Q-Exactive is a hybrid type mass spectrometer that combines quadrupole and orbitrap, a high resolution mass analyzer, and scans a variety of materials at high speed while accurate each material accurately. The mass can be measured with high resolution, as well as quantitative analysis using a quadrupole. Therefore, there is an advantage in that the target component can be accurately quantified with high selectivity from a complex sample such as blood. The elemental composition and the theoretical mass of the detection ion for each component are shown in Table 1 below, and the established final MS analysis conditions are shown in Table 2.

< Experimental Example 1> Derivatization Selection of reagents

Derivatization reagents selected from the present invention methyl bromoacetate (MeBAc), ethyl bromoacetate (EtBAc), isopropyl bromoacetate (iPrBAc), tert -butyl bromoacetate ( tert -Butyl bromoacetate (tBuBAc), phenyl bromoacetate (PhBAc), and benzyl bromoacetate (BzBAc) are alkyl ester compounds of bromoacetic acid. This derivatization reagent is thought to react effectively to primary, secondary, and tertiary amines as well as to carboxylic acid groups depending on the reaction conditions. Therefore, multiple primary derivatization reactions can occur if the chemical structure of the molecule has both primary and secondary amines or both amine and carboxylic acid groups. The derivatization reaction can be influenced not only by the type of derivatization reagent, but also by its concentration. In particular, in the case of multiple derivatization reactions, the concentration of reagents can have a great influence on reactivity, so it was intended to select the optimum derivatives for each component by preparing them at concentrations of 0.05, 0.1, 0.5, 1.0 mol/L for each derivatization reagent.

The derivative product was analyzed using LC-HRMS using a full scan mode (scan range m/z 50~700, resolving power 70,000). Generated by each component by checking the elemental composition and mass error through the MS spectrum of the peak detected by removing the background noise of the LC-HRMS chromatogram and the exact mass. Each derivative was identified.

Derivatization reagents and reaction patterns according to their concentrations were examined using standard solutions (50 μmol/L) of each of 9 types of analysis targets (HCY, DMG, BET, GBB, CAR, TMA, CHL, TML, TMAO). . When various derivatives are produced through multiple derivatization reactions, the most sensitive MS detection sensitivity for each type of derivative product ('M+Deriv','M+2Deriv' type, etc.) is selected as a representative compound for each type, and The most sensitive derivative was selected as the final analyte.

Summarizing the reaction results of 9 target components for each derivatization reagent, the 9 components had the best MS detection sensitivity when the derivatization reagent was 1.0 mol/L. Therefore, in each derivatization reagent, all of the 9 analyte components were compared at the concentration of 1.0 mol/L, which shows the best detection sensitivity, and 6 reaction reagents were finally compared (FIG. 1).

Overall MS sensitivity enhancement was confirmed in the order of EtBAc, iPrBAc, MeBAc, tBuBAc, BzBAc, PhBAc. Therefore, EtBAc was finally selected as a derivatization reagent for 9 components to be analyzed.

< Experimental Example 2> Hydrochloric acid in filter paper Drip Review (On-spot Reaction)

In order to solve the phenomenon in which the recovery of TMA is volatilized in DBS specifically, the hydrochloric acid solution was previously treated before dripping blood on filter paper. In order to confirm the optimum hydrochloric acid concentration required for this, aqueous hydrochloric acid solutions of 0.001, 0.05, 0.01, 0.05, 0.1, 0.5, and 1.0 mol/L having different concentrations were prepared and compared. As a DBS filter paper, a Whatman 903 protein saver card was used.

As a result, as the concentration of the hydrochloric acid aqueous solution increased, the TMA recovery rate gradually improved, and when the concentration of 0.5 mol/L was treated, the recovery rate was about 100% (FIG. 2). In conclusion, the TMA-hydrochloric acid neutralization reaction prevented the loss of TMA in DBS, and it was found that the optimum efficiency was exhibited at 0.5 mol/L hydrochloric acid.

< Experimental Example 3> Sensitivity

Through the optimized derivatization reaction conditions as in Example 1, the degree of MS detection sensitivity improvement of 7 (HCY, DMG, BET, GBB, CAR, TMA, TML) participating in the reaction among the 9 target components was examined. 20 μmol/L DBS was prepared using SA (surrogate analyte) of the analyte in blood, and then the derivatization reaction was performed to determine the peak area of the resulting derivative and the peak area of the raw material that did not undergo the derivatization reaction. It was confirmed by the ratio (derivative/raw material). The results are presented as the average value derived from a total of 3 repeated tests.

As a result, it was found that HCY could be newly detected through derivatization, and the rest was improved by about 1.6-86.3-fold detection sensitivity for each component (Table 3). In particular, TMA has improved sensitivity more than 80 times compared to the raw material, which is reduced during the desolvation process of electrospray ionization (ESI) ionization due to reduced volatility of TMA due to the functional groups introduced by hydrochloric acid treatment and derivatization reaction. It is estimated that the amount of TMA could be minimized. In addition, the increased molecular structure ESI ionization efficiency is another important factor.

< Experimental Example 4> Selectivity

Since the components to be analyzed in this test are all endogenous substances, the method validation was performed using surrogate components that are stable isotope-labeled compounds of each component. Stable isotope-labeled compounds (HCY-d ₄ , DMG-d ₆ , BET-d ₉ , GBB-d ₉ , CAR-d ₃ , TMA-d ₉ , of blank whole blood and 9 target components) DBS was prepared from blood added with CHL-d ₉ , TML-d ₉ , and TMAO-d ₉ ), and 6 kinds of actual blood were selected and tested as target samples. As a result, it was confirmed that an interference peak was not detected around the retention time of the target component in the LC-HRMS chromatogram of DBS prepared with a blood sample as shown in FIG. 3. In addition, it was confirmed from the results of DBS analysis in which 9 types of SA (surrogate analyte) of the target component and an internal standard were added, all 10 target components were separated smoothly without being affected by other interference components.

< Experimental Example 5> Response Factor

Since the endogenous substance cannot secure a blank matrix, it is difficult to develop an analytical method or quantify the target component. In the present invention, a surrogate analyte (SA) method was introduced to overcome this. The SA method is a method for quantifying a desired endogenous substance in an actual biosample after preparing a calibration curve using a stable isotope-labeled compound having the same chemical properties as the actual component AA (authentic analyte) as SA. This is because the chemistry of the authentic analyte (AA) and the surrogate analyte (SA) is the same, so the chromatographic tendency such as retention time, the MS detection reactivity represented by the peak area, and the extraction and derivatization reaction are theoretically the same. It is based. However, if the MS detection response between the two groups is the same, there is no problem, but if the MS detection response is different, correction is necessary, and this correction constant is called a'response factor (RF)'. In this test, the response coefficient was confirmed by using a solution (matrix-matched standard, MMS) to which a standard solution was added after the standard solution and the actual DBS sample were pretreated.

First, the actual components (authentic analyte; HCY, DMG, BET, GBB, CAR, TMA, CHL, TML, TMAO) of the 9 components to be analyzed and the stable isotope labeling compound (surrogate analyte; HCY-d ₄ ) of each of the components , DMG-d ₆ , BET-d ₉ , GBB-d ₉ , CAR-d ₃ , TMA-d ₉ , CHL-d ₉ , TML-d ₉ , TMAO-d ₉ ) The RF value was calculated by confirming the MS detection reaction in the 0.2-100 μmol/L section. When the RF value is 1, it means that the detection reaction of AA and SA is the same, when it is greater than 1, the detection reaction of SA is greater, and when it is less than 1, it can be interpreted that the detection reaction of AA is greater. Finally, the component whose RF value deviated from 5% (0.95-1.05) based on 1 was tried to quantify after being corrected to the RF value according to the following Equation 1.

[Equation 1]

As a result, all the RF values of the 9 components showed consistent results without significant differences depending on the concentration. However, some differences occurred in the RF values depending on the components.The MS detection reaction between the authentic analyte (AA) and the surrogate analyte (SA) is the largest, that is, the component with the lowest RF value at 1 is HCY. The value was shown. The remaining components showed an RF value of 0.88-1.03, so there was no significant difference in the MS detection response between the two groups.

In addition, it was also applied to an actual DBS sample to confirm that there was an effect of the matrix. Since AA (Authentic analyte) is present in actual blood, it was tested at a high concentration of 25, 100 μmol/L that can be clearly distinguished from this, and the difference between the peak area ratio of the sample added with AA and the peak area ratio of the blank was calculated. As a result, even in the MMS sample, no difference occurred in the RF value according to the concentration, and an RF value of 0.77-1.06 was shown for each component. When compared with the standard solution, the difference was -4.5-4.5%, which confirmed that the substrate had little effect on the RF results. Finally, HCY, BET, CAR, TMA, TML, TMAO in which the RF value out of 5% (0.95-1.05) in MMS was corrected and corrected for each RF value 0.77, 0.86, 1.06, 0.88, 0.85, 0.92.

< Experimental Example 6> Parallelism Test (Parallelism Assessment)

A parallelity test was performed to evaluate whether the authentic analyte (AA) contained in the actual sample can be accurately quantified using a surrogate analyte (SA). Prepare a calibration curve with DBS prepared by adding AA to the blood, analyze the DBS prepared by adding water (sample) and authentic analyte (QC sample) to the same blood, and substitute the previously prepared calibration curve and enter AA The endogenous concentration and calculated concentration of were calculated respectively. At this time, the RF value for each component obtained in Experimental Example 3 was used as a correction factor. That is, the detected peak area ratio was multiplied by the RF value of each component, and then substituted into a calibration curve to obtain an accurate concentration of AA (authentic analyte). This was repeated 6 times to evaluate the accuracy and precision of the results. Accuracy was measured as a percentage of the calculated concentration in the QC sample divided by the theoretical concentration (the sum of the endogenous concentration and the spiked concentration), and the precision was the relative standard deviation for 6 replicates for each concentration. It was confirmed by.

As a result, the accuracy for the 9 target components was 86.7-109.7%, and the precision was 4.7-11.0%, which satisfied the allowable range.

< Experimental Example 7> Linearity (Linearity)

In order to prove the linearity, a standard solution of 9 (surrogate analyte) of the target component was added to the blood sample to obtain an appropriate concentration, and DBS for a calibration curve of a known concentration was prepared and analyzed by pretreatment. Concentration range of each analyte in blood (HCY-d ₄ and DMG-d ₆ 0.1-50 μmol/L, BET-d ₉ 0.05-100 μmol/L, GBB-d ₉ 0.02-20 μmol/L, CAR- d ₃ 0.1-100 μmol/L, TMA-d ₉ 0.01-20 μmol/L, CHL-d ₉ 0.025-50 μmol/L, TML-d ₉ 0.1-20 μmol/L, TMAO-d ₉ 0.2-100 μmol /L), all components showed good linearity of coefficient of correlation ( R ² ) of 0.99 or more (FIGS. 4 to 6 ).

< Experimental Example 8> Lower Limit of Quantification; LLOQ )

The minimum quantitative limit means the minimum concentration of the target component that can be accurately quantified using the developed test method. In this test method, the signal-to-noise ratio (S/N ratio) on the LC-MS chromatogram was 10 or more, and the accuracy was set to a concentration that satisfies 20% or less with 80-120% accuracy and relative standard deviation. The minimum limit of quantification for the SA (surrogate analyte) of the 9 components to be analyzed is HCY-d ₄ , DMG-d ₆ , CAR-d ₃ , TML-d ₉ 0.1 μmol/L, BET-d ₉ 0.05 μmol/L, GBB -d ₉ 0.02 μmol/L, TMA-d ₉ 0.01 μmol/L, CHL-d ₉ 0.025 μmol/L, TMAO-d ₉ 0.2 μmol/L were measured, and the concentration was adjusted to the lowest concentration in the calibration curve for each component. Was set.

< Experimental Example 9> Recovery

The recovery rate is the peak area ratio ratio (A/B) of each sample (A) pre-treated by adding a standard concentration of a known concentration to the blood sample and sample (B) pre-treated with a corresponding concentration of the standard solution. ). In order to confirm the recovery rate, each component was repeatedly tested six times at three concentrations (low, medium, and high concentration) within the calibration curve range.

As a result, the recovery rate of 9 target components was found to be 72.3-94.6%, and all tests showed good reproducibility within 10.9% of the relative standard deviation.

< Experimental Example 10> Reproducibility

In order to verify the repeatability, a standard solution of 9 kinds of analyzed components (surrogate analyte) was repeatedly injected into LC-HRMS 6 times, and it was confirmed by the relative standard deviation of the peak area ratio for each component. As a result, the relative standard deviation of 1.8-4.1% was observed for all components, confirming that the device reproducibility was good.

< Experimental Example 11> Stability

In order to evaluate the stability of 9 types of target components, there are four types, standard solution stability, autosampler stability, short-term stability, and long-term stability. Tests were performed on the items. For each stability test, the test result was confirmed by indicating whether the peak area ratio increased or decreased according to each day (time) based on the initial peak area ratio of the analyzed component.

First, as a result of a standard solution stability test to confirm the stability of the standard solution itself, it showed a stability of 92.9-105.8% (FIG. 7). Second, when the analysis time was long, as a result of an autosampler stability test to confirm that the sample pre-treated during that time was stable in an autosampler, it showed a stability of 95.4-103.2% (FIG. 8). Third, since DBS can also deliver samples through postal delivery, it was confirmed that short-term stability was taken into account when exposed to room temperature during these samples. As a result, it showed a stability of 92.6-105.1% (Fig. 9). Fourth, as a result of conducting a long-term stability test to confirm that the DBS sample is stable for a long period of time, a stability of 92.2-105.4% was confirmed (FIG. 10 ). Therefore, it was confirmed that the stability of the target component according to the analysis time, sample transport, and sample storage did not have a large problem overall.

In conclusion, the present invention introduced a chemical derivatization method through prevention of volatilization through pretreatment of hydrochloric acid and treatment with ethyl bromoacetate (EtBAc) as a pretreatment for blood droplets as a DBS pretreatment, and improved detection sensitivity, and surrogate analyte (SA) method. A test method was developed to accurately and simultaneously quantitatively analyze even a small amount of endogenous substances using.

Accordingly, arrhythmia such as myocardial infarction, stroke, thromobosis, heart failure, atrial fibrillation, etc. through an analysis method of various compounds related to TMAO metabolism according to the present invention ) It can be useful for diagnosis and analysis of various cardiovascular diseases.

Claims (7)

(a) hydrochloric acid treatment of filtered blood spots (DBS) filter paper;
(b) dripping blood onto the DBS filter paper;
(c) reacting by adding an internal standard solution and an elution solvent to the blood-dropped DBS filter paper, followed by centrifugation to obtain a supernatant;
(d) reacting by adding ethyl bromoacetate (EtBAc) to the obtained supernatant; And
(e) analyzing the reactants obtained in step (d); Simultaneous quantitative analysis method of trimethylamine oxide compounds in the blood containing. According to claim 1,
The trimethylamine oxide-based compound,
Beta in; (BET betaine), γ- butyl trimethylamine N- oxide (trimethylamine N -oxide; TMAO), trimethylamine (trimethylamine; TMA), carnitine (carnitine;; CAR), choline (choline CHL), betaine (γ-butyrobetaine; GBB), trimethyllysine (TML), homocysteine (HCY), dimethylglycine (dimethylglycine; DMG) method for simultaneous quantitative analysis of trimethylamine oxide compounds in the blood. According to claim 1,
The step of hydrochloric acid treatment of the filtered blood spots (DBS) filter paper,
Method for simultaneous quantitative analysis of trimethylamine oxide compounds in blood, characterized in that hydrochloric acid is treated at a concentration of 0.1 to 1.0 mol/L. According to claim 1,
The step of reacting by adding ethyl bromoacetate (EtBAc),
Method for simultaneous quantitative analysis of trimethylamine oxide compounds in blood, characterized in that EtBAc is treated at a concentration of 0.75 to 1.5 mol/L. According to claim 1,
The step of adding an internal standard solution and an elution solvent to the blood-dried DBS filter paper,
Method for simultaneous quantitative analysis of trimethylamine oxide compounds in the blood, further comprising a dithiothreitol (DTT) reducing agent. According to claim 1,
The step of analyzing the reactants is,
Method for simultaneous quantitative analysis of trimethylamine oxide compounds in blood, characterized by analyzing by chromatography/mass spectrometry. According to claim 1,
As for the analysis method,
Method for simultaneous quantitative analysis of trimethylamine oxide-based compounds in blood, which improves the detection sensitivity by 2 to 86 times.

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