Method of analyzing compound including amine group by using Boc compound

Abstract

Provided is an amine group-derivatized composition including a Boc compound for liquid chromatography-mass spectrometry (LC-MS) analysis, and when the amine group-derivatized composition is used in analysis using reverse-phase LC-MS, it is possible to effectively analyze compounds including an amine group and an amino acid at a low cost in a short time.

Claims

1. A method of analyzing a compound comprising an amine group in a sample, the method comprising: contacting a compound represented by Formula 1 with a sample; producing a Boc-amine derivative by coupling the compound comprising an amine group in a sample with the compound represented by Formula 1; and analyzing the Boc-amine derivative by using liquid chromatography-mass spectrometry (LC-MS), ##STR00003## wherein, in Formula 1, R is selected from hydrogen, deuterium, halogen, a nitro group, a cyano group, a substituted or unsubstituted C.sub.1-C.sub.20 alkyl group, a substituted or unsubstituted C.sub.2-C.sub.20 alkenyl group, a substituted or unsubstituted C.sub.2-C.sub.20 alkynyl group, a substituted or unsubstituted C.sub.1-C.sub.20 alkoxy group, a substituted or unsubstituted C.sub.3-C.sub.20 cycloalkyl group, a substituted or unsubstituted C.sub.2-C.sub.20 heterocycloalkyl group, a substituted or unsubstituted C.sub.3-C.sub.20 cycloalkenyl group, a substituted or unsubstituted C.sub.2-C.sub.20 heterocycloalkenyl group, a substituted or unsubstituted C.sub.8-C.sub.20 cycloalkynyl group, a substituted or unsubstituted C.sub.2-C.sub.20 heterocycloalkynyl group, a substituted or unsubstituted C.sub.6-C.sub.20 aryl group, a substituted or unsubstituted C.sub.6-C.sub.20 aryloxy group, a substituted or unsubstituted C.sub.6-C.sub.20 arylthio group, and a substituted or unsubstituted C.sub.1-C.sub.20 heteroaryl group.

2. The method of claim 1, wherein the producing of a Boc-amine derivative is performed under microwave irradiation for about 30 seconds to about 2 minutes.

3. The method of claim 1, wherein the compound represented by Formula 1 is di-tert-butyl dicarbonate.

4. The method of claim 1, wherein the compound comprising an amine group is an amino acid.

5. The method of claim 1, wherein the analyzing of the Boc-amine derivative by using LC-MS is performed by detecting neutral loss of the compound comprising an amine group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

(2) FIG. 1 is a schematic view of an embodiment of amino acid derivatization and reverse-phase liquid chromatography-mass spectrometry (LC-MS) using a Boc compound;

(3) FIG. 2 shows a representative fragmented ionization pattern of Boc-phenylalanine derivative analyzed by a tandem mass spectrometer using electrospray ionization (ESI) in positive ion mode. The distinct neutral loss (100 Da) is observed in all other Boc-amino acid derivatives;

(4) FIG. 3 shows a representative fragmented ionization pattern of Boc-phenylalanine derivative analyzed by a tandem mass spectrometer using ESI in negative ion mode. The distinct neutral loss (74 Da) is observed in all other Boc-amino acid derivatives;

(5) FIG. 4 is a chromatogram of standard amino acids derivatized using a Boc compound and analyzed by a multiple reaction monitoring (MRM) method;

(6) FIG. 5A is an analytical chromatogram of metabolome components included in a rice extract before performing amine derivatization thereof;

(7) FIG. 5B is an analytical chromatogram of metabolome components included in a rice extract after performing dansylation thereof; and

(8) FIG. 5C is an analytical chromatogram of metabolome components included in a rice extract after performing Boc derivatization thereof.

DETAILED DESCRIPTION

(9) Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description.

(10) The present disclosure will be described in further detail with reference to the following examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

Example 1. Method of Analyzing Boc-Amino Acid Derivative Compound Using Reverse-Phase Liquid Chromatography-Mass Spectrometery (LC-MS)

(11) 1.1. Fragment Ion Analysis of Boc-Amino Acid Derivative Compound (Example of Phenylalanine-Boc Compound)

(12) In a container, 20 microliters (L) of a pure solution of 1.0 micromolar (M) phenylalanine (having a molecular weight of 165) was reacted with a Boc-derivatized composition solution (1 L of 0.1 millimolar (mM) Boc in MeOH+2 L of 1 molar (M) NaOH+177 L of 50% aqueous MeOH). Then, the container was sealed using a silicon cap, and then the reaction mixture was reacted for 1 minute under microwave irradiation.

(13) Next, the derivatized solution was ionized by using a triple quadrupole mass spectrometer (API 4000 QTRAP, available from AB SCIEX) using electrospray ionization (ESI) in positive ion mode, thereby obtaining an ion mass of mass-to-charge ratio (m/z) 288. This detected molecular ion corresponds to a molecular ion of phenylalanine (165)+Boc (100)+Na (23). The molecular ion of m/z 288 collided in the tandem mass spectrometer with an energy of 15 electron-volts (eV), thus being fragmented. FIG. 2 is a mass spectrum showing ions of m/z 232 and m/z 188 produced by a loss of molecular weights of 56 and 44 with respect to the molecular ion of m/z 288. FIG. 3 shows the measurement results of the fragmentation pattern of the phenylalanine-Boc compound solution, which was prepared in substantially the same manner as above, obtained by using a tandem mass spectrometer using ESI in negative ion mode. Here, the detected molecular ion of m/z 264 corresponds to a molecular ion of phenylalanine (165)+Boc (100)H (1). The molecular ion of m/z 264 collided in the tandem mass spectrometer with an energy of 16 eV, thus being fragmented. The fragment ionization mass spectrum of FIG. 3 shows ions of m/z 186 produced by a loss of a molecular weight of 74 with respect to the molecular ion of m/z 264. This example shows fragment ions generated by the neutral loss of an amino acid, that is, phenylalanine. In addition to phenylalanine, upon ionization of all standard amino acid compounds, [M+Boc+Na].sup.+ ions were obtained in the above reaction.

(14) 1.2. Analysis of Boc-Amino Acid Derivative Compound (Example of Mixture of 21 Amino Acids)

(15) In order to verify whether, in addition to phenylalanine, mass spectrometry is applicable to all other kinds of amino acids using a Boc compound, 10 L of the resulting product of 21 amino acids derivatized with a Boc compound (in which the final concentration of each amino acid was 0.1 M) was injected using an automatic sample injector, and then separated through a reverse-phase high-performance liquid chromatography (HPLC) column (available from Waters, UPLC C18, Acquity, 2.1100 mm, 1.7 micrometers (m)). Here, as a mobile phase solvent, each of A: water-soluble 95% water (95:5 v/v, H.sub.2O:ACN) and B: 95% ACN (95:5 v/v, ACN:H.sub.2O) were used. The concentration gradient conditions are as follows.

(16) TABLE-US-00001 TABLE 1 HPLC solvent concentration gradient conditions Velocity of Time A % B % flow (minutes) (95:5 v/v, H.sub.2O:ACN) (95:5 v/v, ACN:H.sub.2O) (mL/min) 0 95 5 600 10 0 100 600 10.1 95 5 600 12 95 5 600

(17) Next, API 4000 QTRAP available from AB SCIEX was used as a Tandem QQQ mass spectrometer for ionization using ESI in negative ion mode, followed by measurement. The multiple reaction monitoring (MRM) conditions for each amino acid are as follows.

(18) TABLE-US-00002 TABLE 2 Amino acid MRM conditions of Tandem QQQ mass spectrometry RT No. ID (min) Q1 Q3 DP EP CE CXP 1 Arginine 1.46 273 199 35 10 16 7 2 Asparagine 1.62 231 157 50 10 12 9 3 Glutamine 1.72 245 171 35 10 12 1 4 Serine 1.87 204 130 25 10 12 9 5 Aspartic acid 2.12 232 158 35 10 16 11 6 Glycine 2.16 174 100 35 10 12 5 7 Glutamic acid 2.26 246 172 30 10 14 7 8 Threonine 2.38 218 144 30 10 12 9 9 Alanine 2.63 188 114 45 10 12 7 10 GABA 2.86 251 177 45 10 10 5 11 Proline 3.19 214 140 70 10 20 7 12 Cysteine 3.21 220 146 25 10 10 9 13 Tyrosine 3.41 280 206 50 10 12 29 14 Methionine d3 3.83 251 177 50 10 12 29 15 Methionine 3.89 248 174 70 10 12 7 16 Phenylalanine 3.92 264 190 35 10 12 7 17 Valine 3.92 216 142 30 10 12 1 18 Histidine 4.4 354 280 55 10 14 11 19 Isoleucine 4.53 230 126 50 10 14 11 20 Leucine 4.62 230 156 25 10 12 9 21 Tryptophan 4.7 303 229 50 10 14 11 22 Lysine 4.88 345 271 60 10 16 29

(19) As a result, all 21 kinds of amino acids included in a bean sample were detected, as shown in FIG. 4. Each signal is marked by a number which represents the corresponding amino acid.

Example 2. Test of Boc-Amine Compound Signal Intensity Versus Boc-Amine Derivatization Reaction Time

(20) Table 3 shows the signal intensity of derivatives detected by the Tandem QQQ mass spectrometer versus the derivatization reaction time of the prepared Boc-amine compound. Referring to Table 3, the reaction time for the Boc-amine derivatization reaction was determined. The derivatization reaction was performed in substantially the same manner as in Example 1.2.

(21) TABLE-US-00003 TABLE 3 The signal intensity of amino acid derivatives versus derivatization reaction time Signal Signal Signal Signal Signal intensity intensity intensity intensity intensity (reacted for (reacted for (reacted for (reacted for (reacted for Amino acid 10 seconds) 30 seconds) 1 minute) 5 minutes) 30 minutes) Histidine 0.42 10.sup.4 0.82 10.sup.4 2.12 10.sup.4 2.02 10.sup.4 1.83 10.sup.4 Serine 0.22 10.sup.4 0.56 10.sup.4 1.32 10.sup.4 1.10 10.sup.4 1.03 10.sup.4 Valine 0.32 10.sup.4 0.81 10.sup.4 2.08 10.sup.4 1.86 10.sup.4 1.61 10.sup.4 Isoleucine 0.49 10.sup.4 0.79 10.sup.4 2.19 10.sup.4 2.07 10.sup.4 1.81 10.sup.4

Example 3. Comparison of Derivatization Reaction for Detecting Amine Compound

(22) 3.1 Preparation of Rice Extract

(23) The Boc compound may selectively produce a derivative compound for any compound containing a primary or secondary amine group. At the same time, use of the Boc compound is advantageous in that the Boc compound may not affect detection of secondary metabolome not including an amine group, while providing information on the whole metabolome. In order to verify this advantage, amine compound detectivity of a dansylation derivatization reaction, which is generally used in detection of an amine compound, and that of a Boc-amine derivatization reaction were compared and analyzed. The comparative analysis was carried out as follows. First, 0.04 grams (g) of a seed was ground into powder, which was then sonicated for 1 hour using 4 milliliters (mL) of 50% water-soluble methanol (50/50, v/v, water/methanol). The resultant was then filtrated by using a 0.2 m filter. The extracted sample was dried, and then re-solubilized using a dimethyl sulfoxide (DMSO) solvent at a concentration of 30 mg/mL, thereby preparing a rice extract sample.

(24) 3.2 Boc Derivatization

(25) Aside from the above process, a Boc compound was dissolved in 100% methanol to prepare a 1.0 M solution. Next, 20 L of the rice extract sample, 4 L of reaction solutions for Boc derivatization, 4 L of NaOH (1 M) Boc compound, and 72 L of 50% aqueous MeOH were added to a glass vial, which was then sealed with a silicon cap. Then, the mixture was reacted under microwave irradiation for 1 minute. Accordingly, a Boc-amine derivatization product of the rice extract was obtained.

(26) 3.3 Dansylation Derivatization

(27) For comparative evaluation, a rice extract sample (30 mg/mL) and a reaction solution generally used in a dansylation derivatization reaction were prepared as follows to carry out the derivatization reaction. First, 40 L of a buffer solution (1.0M NaHCO.sub.3/Na.sub.2CO.sub.3) and 5 L of dansyl-chloride (1.0 M in an ACN solution) were mixed together with 20 L of a rice extract sample and 40 L of 50% aqueous MeOH in a glass vial. The reaction was carried out at a temperature of 60 C. for 60 minutes. Afterwards, the reaction solution was cooled to room temperature. 5 L of a NaOH solution (0.25M) was added to the reaction solution, which was then reacted for 10 minutes. 10 L of formic acid (0.425M) was added thereto to complete the reaction.

(28) 3.4 Analysis of Amine-Derivatized Compound

(29) By using HPLC, derivative compounds including each amine group included in rice were separated according to time, and then ionized by ESI in positive ion mode.

(30) 10 L of the finally completed derivatization solution was separated using HPLC, and components thereof were ionized by ESI in positive ion mode. Here, the column and the solvent concentration gradient conditions were the same as those of Example 2.2. Next, a Q-TOF mass spectrometer (microTOF II, available from Bruker) was used to measure the mass of each amino acid derivative compound using ESI in positive ion mode (in a range of 50 m/z to 800 m/z). Here, the ionization conditions were as follows: capillary voltage of 4,500 Volts (V), nebulizer gas of 0.8 bar, dry gas of 8 L/min, and dry temperature of 180 C.

(31) FIG. 5A is a chromatogram of components included in a rice extract, of which a derivatization reaction for amine compounds has not been performed, as analyzed by LC-MS. FIG. 5B is a chromatogram of components included in a rice extract after performing dansylation of amine compounds. FIG. 5C is a chromatogram of components included in a rice extract after performing a Boc-amine derivatization reaction for amine compounds. Referring to FIG. 5B, in the retention time ranging from 1 minute to 8 minutes after performing the dansylation, signals can be seen which did not appear in FIG. 5A. It was found that these signals were caused by amine compounds due to amine derivatization. However, it was found that most of the secondary metabolome components of the rice extract that appeared in the retention time ranging from 6 minutes to 10 minutes disappeared after performing the dansylation. Referring to FIG. 5C, after performing the Boc-amine derivatization, it was found that new amine compounds appeared, and furthermore, the secondary metabolome components of the rice extract also were detected without being affected by the Boc reaction. Accordingly, it was found that the Boc-amine derivatization reaction is effective in derivatization of components including amine compounds, and furthermore, it was found that the Boc-amine derivatization reaction barely affects dissociation of the secondary metabolome not including amine compounds. In metabolomic analysis, upon analysis of hundreds or more samples according to the current analysis processing capability, amine compounds should be analyzed again separately; however, these results provide a significant improvement in this regard.

(32) As described above, according to one or more embodiments, when LC-MS using a Boc compound is used as a method of analysis, it is possible to analyze a compound including an amine group at a low cost in a short time.

(33) When an amine group-derivatized composition including a Boc compound is used for LC-MS analysis, it is possible to analyze a compound including an amine group at a low cost in a short time.

(34) It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.

(35) While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.