JP2020106494A - Method for quantifying ethylamine in biological sample - Google Patents

Abstract

To provide a method for highly sensitively quantifying ethylamine in a biological sample.SOLUTION: Provided is a method for quantifying ethylamine in a biological sample, which includes the steps of: (i) reacting ethylamine in the biological sample with a derivatizing reagent; (ii) separating the derivatized ethylamine in the biological sample by liquid chromatography; and (iii) analyzing the derivatized ethylamine derived from the biological sample separated in the step (ii) by a mass spectrometer.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting and quantifying ethylamine in a biological sample using a liquid chromatograph-mass spectrometer.

Tea has been used habitually all over the world since ancient times, and is now estimated to be consumed by more than two-thirds of the world's population. This is because there are many kinds depending on the manufacturing method, and thus it is widely used as a favorite product to enjoy the scent and taste, and also has various health promoting effects.

Tea, especially as a main component contained in green tea, polyphenols containing catechin, alkaloids, amino acids, dietary fiber, lipids, odor components, vitamins and minerals, etc. are known, among them, theanine, which is one of the amino acids, is , Has attracted attention as one of the components having a health promoting effect. Theanine has a structure in which the side chain carboxyl group of glutamic acid and an ethylamine form an amide bond. When a human drinks tea, theanine is rapidly absorbed in the body, most of which is hydrolyzed to glutamic acid and ethylamine, but some of which is taken up by red blood cells (Non-Patent Document 1).

Studies on the efficacy of theanine have reported antihypertensive action (Non-Patent Document 2), antidepressant action (Non-Patent Document 3), sleep quality improving action (Non-Patent Document 4), etc. This is a study observing changes in emotions and physiological phenomena that appear upon administration, and theanine in biological samples has not been measured.

The reason why there are almost no reported cases where theanine is measured is that the blood half-life of theanine is very short. According to a report on the pharmacokinetics of theanine after drinking tea, it is considered that most of theanine disappears from the blood within 12 hours after ingestion of tea (Non-patent Document 1), so that there is no delay after ingestion. Need to collect blood. This is presumed to be a barrier in measuring theanine.

Up to now, some analysis methods for theanine have been reported, but all of the methods reported in Patent Document 1 and Patent Document 2 were obtained by mixing a sample extracted with a tea component and a standard solution. This is the method of measuring the sample. In these methods, various substances contained in the biological sample may affect the concentration measurement of theanine, and thus are not suitable for the measurement of theanine in the biological sample. The method reported in Non-Patent Document 1 measures theanine in human plasma, but it is presumed that the theanine cannot be detected in a sample collected 12 hours or more after ingestion of tea due to insufficient sensitivity. It With the recent development of analytical methods, there is a report of an analytical method using a mass spectrometer (MS) capable of high-sensitivity detection (Patent Document 3). It is only a measurement of the obtained sample, and it is a method of simultaneously measuring multiple components, and the sensitivity to theanine is considered to be insufficient.

JP-A-01-240855 JP, 2007-155465, A International Publication No. 2005/116629

Scheid L, Ellinger S, Alteheld B, Herholz H, Ellinger J, Henn T, Helfrich HP, Stehle P (2012) Kinetics of L-theanine uptake and metabolism in healthy participants are comparable after ingestion of L-theanine via capsules and green tea . The Journal of Nutrition 142(12), 2091-2096 Yoto A, Motoki M, Murao S, Yokogoshi H (2012) Effects of L-theanine or caffeine intake on changes in blood pressure under physical and psychological stresses. Journal of Physiological Anthropology 31:28 Hidese S, Ota M, Wakabayashi C, Noda T, Ozawa H, Okubo T, Kunugi H (2017) Effects of chronic l-theanine administration in patients with major depressive disorder: an open-label study.Acta Neuropsychiatrica 29(2), 72-79 Lyon MR, Kapoor MP, Juneja LR (2011) The effects of L-theanine (Suntheanine(R)) on objective sleep quality in boys with attention deficit hyperactivity disorder (ADHD): a randomized, double-blind, placebo-controlled clinical trial . Alternative Medicine Review 16(4), 348-354 Zotou A, Notou M (2012) Study of the naphthalene-2,3-dicarboxaldehyde pre-column derivatization of biogenic mono- and diamines in mixture and fluorescence-HPLC determination. Analytical and Bioanalytical Chemistry 403(4), 1039-1048 Hlabangana L, Hernandez-Cassou S, Saurina J (2006) Determination of biogenic amines in wines by ion-pair liquid chromatography and post-column derivatization with 1,2-naphthoquinone-4-sulphonate. Journal of Chromatography A 1130(1), 130-136 Furst P, Pollack L, Graser TA, Godel H, Stehle P (1990) Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials.Journal of Chromatography A 499(19), 557-569 Parshintsev J, Ronkko T, Helin A, Hartonen K, Riekkola ML (2015) Determination of atmospheric amines by on-fiber derivatization solid-phase microextraction with 2,3,4,5,6-pentafluorobenzylchloroformate and 9-fluorenylmethoxycarbonyl chloride.Journal of Chromatography A 1376, 46-52

For these reasons, direct analysis of theanine in the tea component is not necessarily appropriate as a means for grasping the intake situation of tea and conducting more detailed research on theanine. In addition, there are high barriers for use in clinical tests that are simple and quick.

Therefore, the present inventors focused on ethylamine, which is a metabolite of theanine. The blood half-life of ethylamine is significantly longer than that of theanine, and is detected in blood even 24 hours after the ingestion of tea (Non-Patent Document 1). Therefore, not only in samples taken immediately after ingesting tea, but in samples in which the amount and frequency of tea ingestion are unknown It is considered more desirable to measure ethylamine, which has a long residence time.

According to the reports so far, ethylamine is also contained in cheese (Non-Patent Document 5), wine (Non-Patent Document 6), etc., but the amount of ethylamine contained in them is smaller than that of tea. , It is highly conceivable that there is a positive correlation between the intake of tea and the amount of ethylamine in blood in Japanese people who have a culture of ingesting tea on a daily basis. When studying the relationship between the intake of cheese and wine, not only tea, and the amount of ethylamine in the body, it is expected that a more sensitive analysis method will be required because the amount of ethylamine contained in them is small.

Therefore, a method for measuring ethylamine in a biological sample with ease, speed and high sensitivity is required, but such a method does not exist like theanine. For example, the lower limit of quantification of the human plasma ethylamine analysis method by O-phthalaldehyde derivatization reported in Non-Patent Document 1 and Non-Patent Document 7 is about 5 ng/mL, and it has sufficient sensitivity. Is hard to say. In such assays it is unclear whether ethylamine is truly absent or not detected due to the lack of sensitivity of the assay.

Further, in other analytical methods, the lower limit of quantification of the most sensitive analytical method is 1.72 ng/mL (Non-Patent Document 8), but it is a method for measuring ethylamine in the atmosphere and The measurement of ethylamine is not supported. That is, the currently reported measurement system cannot accurately grasp the concentration of ethylamine in a biological sample and the pharmacokinetics, and as a result, research on theanine does not progress.

Therefore, an object of the present invention is to provide a measurement system capable of achieving the demand for grasping the intake situation of tea or theanine by establishing an analysis method that can easily, rapidly and highly sensitively determine the concentration of ethylamine in a biological sample. It is to be.

In addition, in this specification, measurement is used in the same meaning as detection, analysis, quantification and the like. In addition, the sample is used in the same meaning as the sample.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have added an organic solvent to a biological sample, stirred and centrifuged, and then mixed a part of the supernatant with dansyl chloride and sodium hydrogen carbonate. It was found that ethylamine in a biological sample can be quantified with high sensitivity by measuring a heated and cooled sample with a liquid chromatograph-triple quadrupole mass spectrometer (LC-MS/MS). The present invention has been accomplished based on this finding.

That is, the present invention provides the following:
[1] A method for quantifying ethylamine in a biological sample, comprising:
(I) reacting ethylamine in the biological sample with a derivatization reagent,
(Ii) separating the derivatized ethylamine in the biological sample by liquid chromatography, and
(Iii) a step of analyzing a derivatized ethylamine derived from the biological sample separated in the step (ii) with a mass spectrometer,
Including the method.
[2] The method according to [1], wherein in the step (ii), a mobile phase containing ammonium formate, acetonitrile and formic acid is used.
[3] The method according to [2], wherein the step (ii) includes a step of increasing the concentration of acetonitrile in the mobile phase.
[4] The step (ii) includes a step of increasing the concentration of acetonitrile in the mobile phase from a first acetonitrile concentration to a second acetonitrile concentration,
The first acetonitrile concentration is 0% or more,
The second acetonitrile concentration is 30% to 70%,
The method according to [2] or [3].
[5] The method according to any one of [2] to [4], wherein the step (ii) includes a step of reducing the concentration of ammonium formate in the mobile phase.
[6] The method according to any one of [2] to [4], wherein the step (ii) includes a step of increasing the concentration of formic acid in the mobile phase to 0.03% to 0.065%.
[7] The derivatization reagent is orthophthalaldehyde, fluoresamine, naphthalene-
2,3-dicarboxaldehyde, dansyl chloride, 3,5-dinitrobenzoyl chloride, benzenesulfonyl chloride, 2,3,4,5,6-pentafluorobenzyl chloroformate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethoxycarbonyl chloride, 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, 6-aminoquinolyl-N-hydroxysuccinylimide, 4-fluoro-7. -Nitrobenzofurazan, 4-chloro-7-nitrobenzofurazan, fluorescein isothiocyanate, 7-fluoro-4-(N,N-dimethylaminosulfonyl)benzofurazan and 1,2-naphthoquinone-4-sulfonate The method according to any one of [1] to [6], which is any substance selected from the following.
[8] The method according to any one of [1] to [7], wherein the biological sample is pretreated by a deproteinization method before the step (i).
[9] The mass spectrometer comprises a quadrupole type, a quadrupole tandem type, an ion trap type, an ion trap quadrupole hybrid type, a magnetic field deflection type, a time-of-flight type, and a quadrupole time-of-flight hybrid type. The method according to any one of [1] to [8], which is one of more selected types.
[10] The ethylamine is quantified based on a peak area ratio obtained by dividing the peak area value derived from the ethylamine detected by the mass spectrometer by the peak area value derived from the internal standard substance. The method described in either.

According to the present invention, it is possible to quantify the concentration of ethylamine in a biological sample accurately and with high sensitivity (for example, 0.400 ng/mL) as compared with the conventional analysis method of ethylamine. Furthermore, since rapid measurement is possible (for example, the measurement time is 2.8 minutes per sample), it is possible to measure many samples in a short time.

Further, as will be described later, when measuring a sample whose tea intake state is unknown using the present invention, the number of samples whose measured value is less than the lower limit of quantification due to lack of sensitivity can be significantly reduced.

Thus, for example, in a retrospective cohort study, by investigating the ethylamine concentration in human blood whose tea intake status is unknown and the morbidity of various diseases several years after sample collection, etc. It is expected to be applied to clinical research such as clarifying the efficacy of.

FIG. 1 shows LC-MS/MS of ethylamine (a) dansylated and ethylamine-d3 (b) of an internal standard substance (hereinafter sometimes simply referred to as “IS”) after pretreatment of human serum. It is a figure which shows the chromatogram when it measures by. FIG. 2 shows a chromatogram when dansylated ethylamine was measured by LC-MS/MS after pretreatment of a sample obtained by adding an ethylamine standard solution (corresponding to 0.5 ng/mL in human serum) to human serum. It is a figure. FIG. 3 is a diagram showing a histogram (up to 10 ng/mL) showing ethylamine measurement values of 1000 human serum samples.

Hereinafter, embodiments of the present invention will be described in detail, but the aspect of the method of use is not limited to this.

The present invention is a method for quantifying ethylamine in a biological sample. Specifically, it includes a step of derivatizing ethylamine and IS with a derivatization reagent such as dansyl chloride, and a step of quantifying ethylamine by LC-MS/MS using the solution after derivatization. The present invention preferably further comprises the step of removing contaminants in the biological sample.

[Biological sample]
In the present specification, the biological sample is not limited as long as it is a biological sample that can contain ethylamine. For example, humans, primates such as monkeys, cats such as dogs, artiodactyla such as pigs, and heavy teeth such as rabbits Samples obtained from rodent eyes such as eyes, mice or rats can be used. The type of biological sample is not limited, but includes, for example, whole blood, plasma, serum, saliva, urine, stool, sputum, semen, tears, nasal discharge, vagina, nose, rectum, pharynx and urethral swab, excretion, secretion. Examples thereof include biopsy tissue samples, and specifically, serum, plasma or urine is preferable. In the present invention, the biological sample may be used as it is collected, but those skilled in the art may optionally add a treatment such as dilution with water or physiological saline, homogenization, etc. It can be used and the conditions can be set appropriately.

Hereinafter, a biological sample is pretreated with an organic solvent for deproteinization (protein removal), ethylamine and IS are derivatized with dansyl chloride, and the conditions for measurement by LC-MS/MS are described as an example. The invention is not limited to this.

[Preprocessing]
Contaminants contained in a biological sample cause ion suppression and enhancement (matrix effect) with respect to interfering peaks and signal intensities, so appropriate pretreatment should be carried out before measurement by LC-MS/MS. Is desirable. The pretreatment method is not particularly limited as long as impurities can be removed from the biological sample and the dansylated ethylamine and IS can be measured by LC-MS/MS. For example, as a method for removing impurities from a biological sample, dilution, concentration, freezing, thawing, heating, drying, crushing, suspension, sterilization, removal of solids, deproteinization method, liquid-liquid extraction method or solid-phase extraction method Etc. In the present invention, the pretreatment method is a liquid chromatograph (hereinafter sometimes simply referred to as “LC”), which is described later as an analysis method for ethylamine and IS, MS, various conditions for analysis using them, and impurities. Can be appropriately selected according to the type of The pretreatment method can be performed according to a conventional method. Further, the pretreatment may be carried out by a single method, or may be carried out by appropriately combining a plurality of methods.

As a specific pretreatment method, for example, a deproteinization method can be used. The deproteinization method is not limited, for example, addition of acid such as perchloric acid, trichloroacetic acid or metaphosphoric acid, addition of organic solvent such as acetone, acetonitrile, methanol or ethanol, heating, cooling, ultrafiltration or ultrafiltration. A method such as centrifugation may be used. The deproteinization method may be used alone or in combination of two or more kinds as long as it does not affect the measured value of ethylamine.

In the present invention, the type of deproteinization method can be determined in consideration of the influence on the derivatization reaction described later. For example, water, IS and methanol are added to the collected biological sample, and after stirring and centrifugation, a part or all of the supernatant is collected (step 1). In this step, the protein in the serum is denatured and precipitated by mixing the serum and methanol.

If the recovery rate in the pretreatment is low, the quantitative sensitivity is lowered and the measurement accuracy is also lowered. Further, if the impurities are not sufficiently removed, not only the reaction efficiency of the ethylamine and the derivatizing reagent is lowered, but also the matrix effect is easily exerted. Therefore, a pretreatment method that can achieve both improvement of recovery rate and removal of impurities is preferable.

[Derivatization reaction]
In the present invention, by carrying out the derivatization reaction, it becomes possible to quantify ethylamine in a biological sample with high sensitivity and selectivity. The term derivatization as used herein refers to a step performed to improve the separability of ethylamine, which is an analysis target, and to enhance ionization efficiency and detection sensitivity, and a person skilled in the art can determine the structure and properties of ethylamine. Considering this, the derivatization reagent can be appropriately selected and used.

Since ethylamine has an amino group that is easily protonated in the structure, highly sensitive detection is expected in the positive mode in LC-MS/MS measurement. Generally, in the measurement using the electrospray ionization (ESI) method, the conditions that determine the sensitivity include chromatographic mobile phase pH, salt concentration and organic solvent concentration. It is expected that the acidification of the compound promotes the protonation by the ESI method and increases the sensitivity.

On the other hand, however, when an acidic solvent is used, ethylamine has a demerit that its retention power on a reversed-phase column generally used at the time of separation by chromatography is weakened because of its high polarity. As a result, the separation from impurities in the sample becomes insufficient, and the possibility of being affected by interference peaks and matrix effects increases.

Therefore, the present inventors have found that derivatization of ethylamine enables good quantification. Examples of the derivatizing reagent include orthophthalaldehyde, fluoresamine, naphthalene-2,3-dicarboxaldehyde, dansyl chloride, 3,5-dinitrobenzoyl chloride, benzenesulfonyl chloride, 2,3,4,5,6-pentafluoro. Benzyl chloroformate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethoxycarbonyl chloride, 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, 6-aminoquinolyl-N-hydroxysuccinyl imide, 4-fluoro-7-nitrobenzofurazan, 4-chloro-7-nitrobenzofurazan, fluorescein isothiocyanate, 7-fluoro-4-(N,N-dimethylaminosulfonyl) Although benzofurazan, 1,2-naphthoquinone-4-sulfonate, etc. are known, those skilled in the art, even if they are derivatization reagents other than those mentioned above, the nature of the derivatization reagent and the polarity of ethylamine, molecular weight or The derivatizing reagent can be selected in view of the measurement of chemical properties such as stability (against light, heat, oxygen, pH, etc.).

These derivatization reagents can be used either as pre-column derivatization reagents or post-column derivatization reagents. A derivatizing reagent may be selected.

The case where dansyl chloride is used as the derivatization reagent will be described below as an example, but the present invention is not limited thereto.
As the derivatization reaction step, for example, a dansyl chloride solution and an alkaline reagent or solution are added to the supernatant obtained in step 1 (step 2). As the alkaline reagent to be added, sodium hydrogen carbonate, sodium carbonate, sodium hydroxide, or the like, or a solution obtained by dissolving them may be used. Specifically, sodium hydrogen carbonate is preferred. By adding sodium hydrogen carbonate, the mixed solution becomes alkaline and the dansylation reaction of ethylamine and IS can be promoted. Those skilled in the art can appropriately set the conditions such as the concentration and pH of the alkaline reagent or solution to be added.

The mixed solution obtained in step 2 is heated, allowed to cool, and then stirred (step 3). Upon heating, ethylamine and IS react rapidly with dansyl chloride to produce dansylated ethylamine and IS. The reaction solution obtained in step 3 can be analyzed by LC-MS/MS.

[analysis]
The composition of LC and MS is not particularly limited as long as dansylated ethylamine and IS are separated from contaminants by LC and can be measured by MS. LC and MS may be connected in series with each other or may be independent from each other. As an apparatus used in the method of the present invention, for example, LC-MS/MS configured by connecting LC and MS in series can be used. By using LC-MS/MS, the dansylated ethylamine and IS separated by LC can be subsequently analyzed by MS.

[Separation process]
In the method of the present invention, the dansylated ethylamine obtained in step 3 and impurities containing an amino functional group-containing compound other than ethylamine derived from the dansylated biological sample are separated by liquid chromatography (step 4 ).

The LC preferably includes a liquid feed pump, a degassing unit, an autosampler, and a column oven, and may be connected to a system controller or a detector. As the LC, for example, a high performance liquid chromatograph (HPLC) can be used. Separation conditions by HPLC can be appropriately set by those skilled in the art based on the description in this specification and ordinary methods. As the LC, an ultra high performance liquid chromatograph (UHPLC) that can perform separation analysis more rapidly and with higher sensitivity than HPLC may be used. The separation condition by UHPLC can be performed in the same manner as the examination when setting the condition of HPLC, and those skilled in the art can set the condition as appropriate.

The mobile phase used for chromatography is not particularly limited as long as dansylated ethylamine and IS are separated from impurities and can be measured by MS. For example, it is possible to keep the pH constant, it is stable even when mixed with an organic solvent, it does not cause unnecessary reaction with sample components, and the stationary phase and equipment of the column such as cleavage of chemically modified groups and dissolution of carriers. It is preferable that the solution does not deteriorate or that non-volatile salts do not interfere with the measurement by MS. These solutions may be used alone or in combination of two or more kinds. Good.

The composition of the mobile phase, the salt concentration, and the pH can be appropriately set according to various conditions such as the properties of the dansylated ethylamine and IS, the type of impurities, and the like. For example, the mobile phase composition, salt concentration and pH are preferably set to enhance the ionization efficiency of the dansylated ethylamine. It is also possible to set the composition of the mobile phase, the salt concentration and the pH using an ion count which indicates the ionized proportion. Further, the composition of the mobile phase, the salt concentration and the pH are preferably set so that the dansylated ethylamine and IS are not affected by interference peaks and matrix effects.

As the mobile phase, it is preferable to use, for example, two kinds of liquid A (aqueous system) and liquid B (organic solvent system). The liquid A may contain ammonium formate, ammonium acetate, ammonium hydrogen carbonate, ammonium hydroxide, formic acid, acetic acid, trifluoroacetic acid, triethylamine, tetrahydrofuran, an organic solvent, or the like. Specifically, an ammonium formate aqueous solution is preferable. As the liquid B, for example, methanol, ethanol, propanol, isopropanol, acetonitrile or the like may be used, and ammonium formate, ammonium acetate, ammonium hydrogen carbonate, ammonium hydroxide, formic acid, acetic acid, trifluoroacetic acid, triethylamine, tetrahydrofuran. , Or water may be included. Specifically, acetonitrile/formic acid is preferable.

Hereinafter, an example will be described in which an aqueous solution of ammonium formate is used as the solution A (aqueous system) and acetonitrile/formic acid is used as the solution B (organic solvent system), but the present invention is not limited to this mode.
When two or more types of mobile phases are used in combination, the content rate of each mobile phase with respect to the total flow rate may be appropriately changed. The rate of change of the ratio may or may not be constant. Further, the ratio may be repeatedly increased and decreased. Acetonitrile and formic acid may or may not be contained in the mobile phase throughout the separation process. The change may be a continuous change (gradient), an intermittent change (stepwise), or a combination thereof. Gradient or stepwise conditions can be appropriately set according to various conditions such as properties of dansylated ethylamine and IS, and types of impurities.

The flow rate of the mobile phase can be set in the range of 0.1 to 1.5 mL/min, for example. Specifically, 0.2 to 1.0 mL/min is preferable, and 0.7 mL/min is more preferable.

The gradient conditions can be set, for example, as the conditions described below. That is, a gradient can be applied to the acetonitrile concentration so that the acetonitrile concentration (v/v%) in the mobile phase gradually increases from the first concentration (M1) to the second concentration (M2). M1 and M2 can be appropriately set according to various conditions such as properties of dansylated ethylamine and IS, types of contaminants, and the like. M1 may be, for example, 0% or more, 10% or more, or 20% or more, and may be less than 30%. M2 may be, for example, 30% or more or 40% or more, and 70% or less or 65% or less. Specifically, for example, a gradient may be applied to the acetonitrile concentration so that the acetonitrile concentration in the mobile phase gradually increases to 30 to 65%. The rate of change of acetonitrile concentration may or may not be constant. The acetonitrile concentration may be repeatedly increased and decreased before changing from M1 to M2.

The acetonitrile concentration may change further after reaching M2. For example, the acetonitrile concentration may further increase, decrease, or increase and decrease after reaching M2. For example, the acetonitrile concentration may be repeatedly increased and decreased after reaching M2 and before changing to M1 again. Specifically, for example, after reaching M2, it may be increased to 95% and continuously decreased to 30%.

In addition, for example, gradient conditions can be appropriately set in order to avoid duplication of elution time with dansylated dimethylamine and the like, which may affect the measurement of dansylated ethylamine and IS. ..

The separation step may include a step of increasing or decreasing the concentration of formic acid (v/v%) in the mobile phase. That is, for example, a gradient can be applied to the formic acid concentration so that the concentration of formic acid in the mobile phase gradually increases from the first concentration (A1) to the second concentration (A2). A1 and A2 can be appropriately set according to various conditions such as properties of dansylated ethylamine and IS, types of contaminants, and the like. A1 and A2 can be set within the range of 0 to 0.1%, for example. Specifically, for example, a gradient may be applied to the formic acid concentration so that the formic acid concentration in the mobile phase gradually increases to 0.03 to 0.065%. The rate of change of formic acid concentration may or may not be constant. The formic acid concentration may be repeatedly increased and decreased. The formic acid concentration may change further after reaching A2. For example, the formic acid concentration may further decrease, increase, or increase/decrease after reaching A2.

The concentration gradient can mix two or more solutions having different compositions with varying ratios. The combination of solutions can be appropriately selected so that a desired gradient is formed.

The gradient may be applied to both acetonitrile and formic acid concentrations together. For example, by mixing a first solution containing formic acid and not containing acetonitrile and a second solution containing acetonitrile and not containing formic acid while changing the ratio, a gradient can be obtained for both concentrations of acetonitrile and formic acid. You can call. Examples of the first solution include formic acid itself and an aqueous solution containing formic acid. Examples of the second solution include acetonitrile itself and an acetonitrile-containing aqueous solution.

When preparing a mobile phase by mixing two or more solutions, the concentration of acetonitrile and formic acid in the two or more solutions is the concentration of acetonitrile and formic acid in the mixed mobile phase as exemplified above. The concentration of acetonitrile and formic acid in the mobile phase can be appropriately set according to the mixing ratio. For example, when a formic acid-containing aqueous solution is mixed with acetonitrile and used as a mobile phase, the formic acid concentration (v/v%) in the formic acid-containing aqueous solution may be 0.05% or more, or 0.1% or more, and 0. It may be 4% or less or 0.2% or less. Specifically, it may be, for example, 0.1 to 0.2%.

The separating step may include reducing the concentration of ammonium formate (mM) in the mobile phase. That is, for example, a gradient can be applied to the ammonium formate concentration so that the ammonium formate concentration in the mobile phase gradually decreases from the first concentration (B1) to the second concentration (B2). B1 and B2 can be appropriately set according to various conditions such as properties of dansylated ethylamine and IS, types of impurities, and the like. B1 and B2 can be set within a range of 1 to 10 mM, for example. Specifically, for example, a gradient may be applied to the ammonium formate concentration so that the ammonium formate concentration in the mobile phase gradually decreases to 3.5 to 7.0 mM. The rate of change of ammonium formate concentration may or may not be constant. The ammonium formate concentration may be repeatedly increased and decreased. The ammonium formate concentration may be further changed after reaching B2. For example, the formic acid concentration may further decrease, increase, or increase/decrease after reaching B2.

The separation column used for chromatography is not particularly limited as long as dansylated ethylamine and IS are separated from contaminants and MS can be measured. For example, a filler having a C30 alkyl chain chemically bonded to a silica gel carrier, a filler having an octadecyl group (C18) chemically bonded, a filler having an octyl group (C8) chemically bonded, and a butyl group (C4) chemically bonded. A column such as a packing material or a packing material in which a phenyl group (Ph) is chemically bonded can be used. Specifically, a column using a packing material in which C18 is chemically bonded may be used. The particle size of the filler may be 5.0 μm or less, preferably 2.0 μm or less, and more preferably 1.8 μm. The length of the column may be 300 mm or less, preferably 150 mm or less, and more preferably 50 mm. The inner diameter of the column may be 10 mm or less, preferably 4.6 mm or less, and more preferably 2.1 mm.

A guard column may be connected to the inlet side of the separation column in order to protect the separation column from contaminants. The guard column preferably has the same packing and inner diameter as the separation column and a length shorter than the separation column, but is not limited thereto.

The column oven may heat the separation column and the guard column. The set temperature can be appropriately set according to the separation of dansylated ethylamine and IS and impurities, the type of mobile phase, the flow rate, the pressure applied to the measuring instrument, and the like. For example, it may be 20 to 60°C, and specifically 30°C.

[Analysis process]
In the method of the present invention, the derivatized ethylamine derived from the biological sample separated in step 4 is analyzed with a mass spectrometer (step 5). Those skilled in the art can appropriately set the measurement conditions by MS on the basis of the description in this specification and ordinary methods.

The MS can be any known MS such as a magnetic field deflection type, a quadrupole type, an ion trap type, a time-of-flight type, or a hybrid type of these, and specifically, a triple quadrupole mass spectrometer. Is more preferable. The triple quadrupole mass spectrometer is an MS equipped with an ion source, a quadrupole (Q1), a collision cell, a quadrupole (Q3) and a detector. The substance to be measured is ionized by the ion source and becomes a precursor ion. Only ions having a specific mass-to-charge ratio (m/z) pass through Q1 and collide with an inert gas such as argon filled in the collision cell to generate product ions. Of the produced product ions, only ions of a specific m/z pass through Q3 and are detected by the detector. Examples of the ionization method include an ESI method, an atmospheric pressure chemical ionization (APCI) method, a fast atom bombardment (FAB) method, an atmospheric pressure photoionization (APPI) method, and a sonic ionization (SSI) method, and the ESI method is particularly preferable. .. The measurement mode includes a positive mode and a negative mode, and the positive mode is particularly preferable. In the positive mode of the ESI method, a proton adduct, an ammonium adduct, a sodium adduct, a potassium adduct, or the like is generated, and it is particularly preferable to detect the proton adduct. Scan types include multiple reaction monitoring (MRM) and selected ion monitoring (SIM), with MRM being particularly preferred.

When dansylated ethylamine and IS are ionized in a positive mode using a triple quadrupole mass spectrometer and measured by the ESI method and MRM, the precursor ion can select a proton adduct. Specifically, the dansylated ethylamine and IS precursor ions are preferably set to 279.1±0.5 and 282.1±0.5, respectively. The product ion of dansylated ethylamine is 115.1±0.5, 128.1±0.5, 156.1±0.5, 157.1±0.5, or 264.1±0.5. Etc. can be used. The product ion of the dansylated IS is 115.1±0.5, 156.1±0.5, 157.1±0.5, 252.1±0.5, or 264.1±0.5. Etc. can be used. Specifically, it is preferable to set both dansylated ethylamine and IS to 156.1±0.5.

Other setting conditions include Dwell time, Q1 PreBias, collision energy (CE), Q3 PreBias, Q1 resolution (Q1 Resolution), Q3 resolution (Q3 Resolution), nebulizer gas and its flow rate, heating gas and its flow rate, interface temperature. , Desolvation line temperature, heat block temperature, drying gas and its flow rate, collision induced dissociation (CID) gas and its gas pressure, and the like can be appropriately set by those skilled in the art. The conditions that can be used in the present invention include those having different names depending on the type of measuring equipment.

In the present invention, the substance to be measured can also include analogs in which ² H, ¹³ C or ¹⁵ N isotope is introduced into a part or all of hydrogen, carbon and nitrogen of ethylamine. An external standard method or an internal standard method can be used for the quantification of these compounds. There is no particular limitation as long as the substance to be measured can be quantified, but it is preferable to use the internal standard method because an error in injection amount, pretreatment operation or measurement can be prevented. For example, in the chromatogram obtained by mass spectrometry, the ethylamine can be quantified based on the peak area ratio obtained by dividing the peak area value derived from ethylamine by the peak area value derived from the internal standard substance.

IS is a component that is not contained in the sample, and is preferably a compound that can be completely separated from components that cause interference peaks and matrix effects and that has properties and a structure similar to the substance to be measured. For example, an analog of ethylamine can be used, and specifically, ethylamine-d3 is more preferable. Those skilled in the art can appropriately select the IS to be used.

Hereinafter, the present invention will be described in detail with reference to Examples, but these do not limit the scope of the present invention.

<<Example 1: LC-MS/MS conditions>>
All quantitative experiments were performed with LCMS-8050 (manufactured by Shimadzu Corporation) using an ESI unit as an ion source. The LC is composed of a system controller (CBM-20A), two liquid feed pumps (both LC-30AD), a degassing unit (DGU-20A5R), an autosampler (SIL-30ACMP) and a column oven (CTO-20AC). The system was used. This system is equipped with LabSolutions LCMS version.
Controlled with 5.82 SP1.

ACQUITY UPLC HSS T3 Column (50 mm×2.1 mm id, 1.8 μm) as a separation column and ACQUITY UPLC HSS T3 VanGu as a guard column were used to separate the dansylated ethylamine and IS from impurities.
An ard Pre-Column (5 mm×2.1 mm id, 1.8 μm) column (all manufactured by Waters) was used, and the column temperature was set to 30° C.

As the mobile phase, a 10 mM ammonium formate aqueous solution was used for the solution A, and acetonitrile/formic acid (1000/1, v/v) was used for the solution B. The flow rate of the mobile phase was 0.7 mL/min, and the gradient conditions were as follows: 0% to 2.20 minutes, 30% B to 65% B; 2.20 to 2.21 minutes, 65% B to 95%. B; 2.21 to 2.70 minutes, 95% B uniform; 2.70 to 2.71 minutes, 95% B to 30% B; 2.71 to 2.80 minutes, 30% B uniform; It was set like.

The needle washing solvent used was methanol/water (1/1, v/v).

The autosampler was set at 4° C. and the sample injection amount was 1 μL. The measurement mode was set to the positive mode, and dansylated ethylamine and IS were measured by MRM.

Various parameters of MS are as follows: nebulizer gas flow rate (nitrogen gas), 3 L/min; heating gas flow rate (air), 10 L/min; drying gas flow rate (nitrogen gas), 10 L/min; interface temperature, 400° C.; heat Block temperature, 400° C.; desolvation line temperature, 250° C.; CID gas (argon gas) pressure, 270 kPa;

Ethylamine-d3 was used as the internal standard substance. Various parameters of dansylated ethylamine (DNS-EA) and IS (DNS-IS) were set as shown in Table 1, respectively.

<<Example 2: Preparation of standard solution>>
A stock solution of ethylamine and IS (1 mg/mL) was prepared in water. A stock solution of ethylamine was serially diluted with water to prepare ethylamine standard solutions with concentrations of 2, 5, 10, 25, 50, 125, 200 and 250 ng/mL. The IS stock solution was similarly diluted with water to prepare a 20 ng/mL IS standard solution. Ethylamine standard solutions of 5, 50 and 200 ng/mL were also used for the preparation of quality control samples for measurement. These standard solutions were stored at 4°C until use, and were returned to room temperature at the time of use.

<<Example 3: Pretreatment operation and measurement>>
50 μL of human serum (50 μL of water for preparation of calibration curve sample and quality control sample) was dispensed into a polypropylene tube of 1.5 mL, and 10 μL of water (2, 5, 10, 25, 50, 125 for preparation of calibration curve sample) , 200 and 250 ng/mL of ethylamine standard solution, 10 μL of 5, 50 and 200 ng/mL of ethylamine standard solution at the time of quality control sample preparation, 10 μL of IS standard solution (20 ng/mL) and 200 μL of methanol were added. After stirring for 5 seconds with a mixer, the mixture was centrifuged at 4° C. at 20,000×g for 3 minutes, and 150 μL of the supernatant was transferred to another 1.5 mL polypropylene tube.

To this tube, 75 μL of 0.1 M sodium hydrogen carbonate aqueous solution and 150 μL of 2 mg/mL dansyl chloride acetone solution were added, and ethylamine and IS were dansylated by heating at 65° C. for 30 minutes. After the reaction solution was allowed to cool to room temperature, the whole amount was transferred to a measurement vial.

From this vial, 1 μL was injected into LC-MS/MS to measure dansylated ethylamine and IS. After pretreatment of human serum, a chromatogram of dansylated ethylamine and IS measured by LC-MS/MS is shown in Fig. 1. Ethylamine standard solution (corresponding to 0.5 ng/mL in human serum) was added to human serum. The chromatogram when the dansylated ethylamine was measured by LC-MS/MS after pretreatment of the prepared sample is shown in FIG.

A straight line obtained by linearly regressing the peak area ratio of dansylated ethylamine to dansylated IS with respect to the concentration of added ethylamine was used as a calibration curve to quantify ethylamine in human serum. The lower limit of quantification of the calibration curve was 0.400 ng/mL, and a weighting of 1/X ² (X: concentration of ethylamine in human serum) was used.
The ethylamine in serum can be accurately measured by the method for determining ethylamine in human serum constructed according to the present invention.

<<Example 4: Quantitative results of ethylamine in human serum>>
FIG. 3 shows a histogram of ethylamine measurement values obtained when 1000 human serum samples, which were previously collected and frozen, were measured using the method for quantifying ethylamine in human serum constructed according to the present invention. As a result, the ethylamine concentration could be quantified in 910 samples, which is 91.0% of the whole.

In addition, the breakdown of the measured ethylamine concentration is as follows: 190 cases (19.0%) in the concentration range of 0.400 to 1.00 ng/mL, and 191 cases (19 cases in the concentration range of 1.01 to 2.00 ng/mL). 0.1%), 121 cases with a concentration range of 2.01 to 3.00 ng/mL (12.1%), 86 cases with a concentration range of 3.01 to 4.00 ng/mL (8.6%) and 4 The concentration range of 0.01 to 5.00 ng/mL was 60 distributions (6.0%).

There are 738 specimens in which the concentration of ethylamine in human serum is 5.00 ng/mL or less, which corresponds to 73.8% of the whole. When the conventional method for analyzing ethylamine in biological samples was used, it was revealed that the amount of these samples was less than the lower limit of quantification, and the intake status of tea or theanine could not be grasped.

According to the present invention, the concentration of ethylamine present in a serum sample or the like can be easily, quickly and highly sensitively determined. This could provide new insights in a retrospective cohort study investigating tea habits and the prevalence of various diseases. The use of the present invention is not limited to this, and is expected to contribute to research for investigating the efficacy of tea.

Claims (10)

A method for quantifying ethylamine in a biological sample, comprising:
(I) reacting ethylamine in the biological sample with a derivatization reagent,
(Ii) separating the derivatized ethylamine in the biological sample by liquid chromatography, and
(Iii) a step of analyzing a derivatized ethylamine derived from the biological sample separated in the step (ii) with a mass spectrometer,
Including the method. The method according to claim 1, wherein in the step (ii), a mobile phase containing ammonium formate, acetonitrile and formic acid is used. The method of claim 2, wherein step (ii) comprises increasing the concentration of acetonitrile in the mobile phase. Said step (ii) comprises increasing the concentration of acetonitrile in the mobile phase from a first acetonitrile concentration to a second acetonitrile concentration,
The first acetonitrile concentration is 0% or more,
The second acetonitrile concentration is 30% to 70%,
The method according to claim 2 or 3. 5. The method of any one of claims 2-4, wherein step (ii) comprises reducing the concentration of ammonium formate in the mobile phase. 5. The method according to any one of claims 2-4, wherein step (ii) comprises increasing the concentration of formic acid in the mobile phase to 0.03% to 0.065%. The derivatization reagent is orthophthalaldehyde, fluoresamine, naphthalene-2,3-dicarboxaldehyde, dansyl chloride, 3,5-dinitrobenzoyl chloride, benzenesulfonyl chloride, 2,3,4,5,6-pentafluorobenzyl. Chloroformate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethoxycarbonyl chloride, 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, 6 -Aminoquinolyl-N-hydroxysuccinyl imide, 4-fluoro-7-nitrobenzofurazan, 4-chloro-7-nitrobenzofurazan, fluorescein isothiocyanate, 7-fluoro-4-(N,N-dimethylaminosulfonyl) The method according to any one of claims 1 to 6, which is any substance selected from the group consisting of benzofurazan and 1,2-naphthoquinone-4-sulfonate. The method according to claim 1, wherein the biological sample is pretreated by a deproteinization method before the step (i). The mass spectrometer is selected from the group consisting of a quadrupole type, a quadrupole tandem type, an ion trap type, an ion trap quadrupole hybrid type, a magnetic field deflection type, a time-of-flight type, and a quadrupole time-of-flight hybrid type. 9. The method according to any one of claims 1-8, which is of any type. The ethylamine is quantified based on a peak area ratio obtained by dividing the peak area value derived from the ethylamine detected by the mass spectrometer by the peak area value derived from the internal standard substance. 10. The method described.

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