JP2873040B2 - Derivatization reagent for high-performance liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to high performance liquid chromatography (hereinafter referred to as HPLC).
Abbreviated. )), A new and effective derivatization (labeling) reagent.

[Background of the Invention]

Carboxylic acids are widely distributed in nature and play an important role as nutrients and metabolites in living organisms.

Since the carboxyl group is an extremely weak chromophore, it is necessary to derivatize (label) this compound in order to detect a compound having a carboxyl group with high sensitivity unless otherwise having a strong chromophore. .

At present, of the detection methods used in HPLC, the fluorescence detection method is one of the most excellent methods in terms of selectivity and sensitivity. Various compounds have been developed as fluorescent labeling agents for quantifying carboxylic acids by HPLC. For example, 4-bromomethyl-7-methoxycoumarin, 1-bromoacetylpyrene, 9-aminophenanthrene, 9-anthryldiazomethane, 3-bromomethyl-6,7-dimethoxy-1-methyl-2 (1H) -quinoxalinone and the like. This is a typical example. However, since all of these labeling agents have low reactivity with carboxyl groups, complete derivatization of carboxylic acid requires a long time, a high reaction temperature, or both. To either. Performing the reaction at a high temperature for a long time not only complicates the derivatization operation but also clearly causes a problem when quantifying heat-sensitive substances such as α-ketocarboxylic acid. Also, 9-anthryldiazomethane and the like have a problem in terms of reagent stability.

In contrast, Ingalls et al. (ST Ingalls et al., J. Chro
matogr., 299 , 365 (1984)] has developed 4'-bromophenazyl trifluoromethanesulfonate as a highly reactive UV-labeling agent for derivatizing carboxylic acids. This reagent has a great feature in that carboxylic acids can be completely derivatized to the corresponding derivative within 5 minutes at room temperature in the presence of N, N-diisopropylethylamine in acetonitrile, but it is still not sensitive. There is still room for improvement and there is still dissatisfaction with the fact that only UV detection is possible and fluorescence detection is not possible.

On the other hand, some of the present inventors have reported that 2- (phthalimino) ethyl trifluoromethanesulfonate (hereinafter abbreviated as PE-OTf) as a novel UV labeling agent having high reactivity with carboxylic acids and high detection sensitivity. Has been developed and has been presented earlier (The 58th Annual Meeting of the Chemical Society of Japan). Although this reagent is an excellent carboxylic acid labeling agent that has never been seen before, it has a problem in that the stability of the reagent is poor and storage at room temperature is impossible.

[Object of the invention]

The present invention has been made in view of the situation as described above, has high reactivity with carboxylic acid, is applicable to both UV detection and fluorescence detection, and has high detection sensitivity, and is excellent in reagent stability, An object of the present invention is to provide a novel labeling agent that can be stored for a long time even at room temperature.

[Summary of the Invention]

The present invention provides a compound represented by the general formula [I]: (Wherein, R ^{1 to} R ⁶ each independently represent a hydrogen atom, a lower alkyl group, a lower alkoxy group, or a halogen atom;
R ³ and R ⁴ may combine with each other to form an aromatic ring, and R represents a hydrogen atom or a lower alkyl group;
n represents an integer of 0 to 10. The present invention relates to a derivatization reagent for high performance liquid chromatography, comprising the compound represented by the formula (1).

Also, the present invention relates to the formula (Hereinafter abbreviated as NE-OTf) and a compound represented by the formula (Hereinafter abbreviated as OPNE-OTf).

In the general formula [I], R ^{1 to} R ⁶ represent a hydrogen atom,
For example, a lower alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group and an amyl group (which may be linear or branched), for example, a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and an amiloxy group A lower alkoxy group such as a group,
Or a halogen atom such as chlorine, bromine or fluorine; and R represents a hydrogen atom or a lower alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group or an amyl group (linear or branched). Any of them is possible).
Is an integer of 0 to 10. When R is a lower alkyl group, the carbon atom bonded to R is, of course, an asymmetric carbon atom. Becomes

The compound represented by the general formula [I] according to the present invention can be easily synthesized, for example, by the following synthesis route.

That is, first, substituted or unsubstituted 2,3-naphthalenedicarboxylic anhydride ([II]) and amino alcohols ([II]) according to the method described in Org. Syn., Col. Vol. 5 , 973 (1973) and the like. [II
I]) in a non-polar solvent (preferably dried) which can azeotrope with water such as benzene, toluene and xylene.
Reflux and dehydrate for ~ 5 hours. After the reaction, the reaction solution is cooled to remove the product, washed with water, and if necessary, recrystallized using a suitable recrystallization solvent such as ethanol to obtain 2,3-naphthaliminoalkanols ([IV] ) Is obtained in high yield.

Then, the 2,3-naphthaliminoalkanols ([IV]) are halogenated with, for example, chloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride or the like in the presence of an organic base such as pyridine or triethylamine. Reaction with trifluoromethanesulfonic anhydride in an organic solvent such as hydrocarbons or diethyl ether at low temperature, preferably at 0 ° C or lower, more preferably at -5 ° C or lower for 0.5 to 5 hours. After the reaction, the reaction solution is sufficiently washed with water, dried over anhydrous magnesium sulfate and the like, the solvent is distilled off, and the obtained residue is subjected to a suitable recrystallization solvent such as n-hexane, n-heptane, dichloromethane or the like, or The desired 2,3-naphthaliminoalkyltrifluoromethanesulfonates ([I]) of the present invention can be easily obtained by recrystallization once or several times using a mixed solvent of dichloromethane and carbon tetrachloride.

In addition, the 2,3-naphthaliminoalkanols represented by the general formula [IV] can be produced by, for example, the general formula [II]
Is reacted with ammonia or ammonium carbonate to give a substituted or unsubstituted 2,3-naphthalenedicarboxylic acid imide, which is then potassium carbonate, sodium carbonate, After treatment with an alkali such as lithium carbonate to form an alkali metal salt, the formula (In the formula, X represents a halogen atom, and R and n are the same as those described above.).

The derivatization reagent according to the present invention can be used particularly effectively for derivatization of fatty acids. That is, by derivatizing fatty acids to be separated and quantified by HPLC into fatty acid esters using the derivatization reagent according to the present invention, a formula having strong UV absorption and fluorescence can be obtained. By introducing a group represented by the formula (1) and labeling with UV and fluorescent, and then subjecting it to HPLC, these fatty acids can be fractionated and quantified with extremely high sensitivity.

The derivatization reaction using the derivatization reagent according to the present invention comprises:
For example, it can be easily implemented as follows.

That is, fatty acids to be separated and quantified by, for example, HPLC are converted to an appropriate solvent (for example, acetonitrile, acetone,
Dissolved in dioxane and the like, and to this are added, for example, a reaction accelerator such as 18-crown-6 and a suitable neutralizing agent (for example, potassium fluoride, potassium carbonate, dipotassium phosphate, etc.). If a derivatizing reagent is added and the mixture is stirred and reacted at room temperature for a certain period of time, all of the fatty acids are completely esterified (the reaction is usually completed within 10 minutes). The amount of 18-crown-6 or the like to be used is desirably equivalent to or more than the compound represented by the general formula [I], and when too small, the derivatization yield decreases. The amount of the neutralizing agent is desirably an amount capable of neutralizing all the acids present in the system. The amount of the compound represented by the general formula [I] is desirably about 5 times or more the molar concentration of the fatty acid to be derivatized. After the completion of the reaction, the supernatant is taken and subjected to HPLC, whereby these fatty acids can be separated and quantified with extremely high sensitivity.

The derivatization reagent according to the present invention is characterized in that it can be applied to both UV detection and fluorescence detection. Detection limit (S / N =
3) In the case of NE-OTf, for example, 100 fmol (λmax
= 259 nm, εmax = 62,000), 4 fmol (λex = 25
9 nm, λem = 394 nm), and the linear regions are 10 ⁻⁸ to 10 ^{−4 respectively.}
M and 10 ^-9 to 10 ^-6 M. However, in the case of PE-OTf, almost no fluorescence is emitted and only UV detection is possible, and its detection limit (S / N = 3) is 200 fmol (λmax = 219 nm, εmax = 42,000).
It is.

All of the compounds represented by the general formula [I] have excellent reagent stability except for those having optical activity, and can be stored usually at room temperature under light shielding for 6 months or more (in the case of PE-OTf, it cannot be stored at room temperature) .). In solution, it depends on the solvent,
For example, it can be stored in acetonitrile at -20 ° C or lower for about one week.

In the compound of the formula [I], in which R is a lower alkyl group, the carbon atom bonded to R is an asymmetric carbon atom, so that a chiral carboxylic acid is derivatized to its diastereomer. This is particularly useful as a reagent for separating optical isomers.

In the case where some of R ^{1 to} R ^{6 in} the general formula [I] are lower alkoxy groups such as methoxy group and ethoxy group, the fluorescence intensity is increased. More preferred.

Increasing the value of n in the general formula [I], that is, increasing the length of the methylene chain increases the HPLC of the derivatized compound.
The retention time at the time increases accordingly. That is, the retention time of derivatized HPLC can be appropriately adjusted by variously changing the value of n in the general formula [I].

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by these Examples.

〔Example〕

Example 1.2 Synthesis of 2- (2,3-naphthalimino) ethyltrifluoromethanesulfonate (NE-OTf) (1) Synthesis of 2- (2,3-naphthalimino) -1-ethanol 2,3-naphthalene in 300 ml of dry toluene 9.9 g (0.05 mol) of dicarboxylic anhydride and 3.1 g of 2-aminoethanol
(0.05 mol), and the mixture was subjected to a dehydration reaction for 3 hours under heating and reflux. After completion of the reaction, the reaction solution was cooled to remove the product, washed three times with 50 ml of cold water three times, and recrystallized with ethanol to obtain 10 g of the desired naphthalimide transparent needles. yield
83%.

mp 192-195 ° C.

Elemental analysis: C ₁₄ H ₁₁ NO ₃ Calculated (%): C, 69.71; H, 4.56; N, 5.81.

Found (%): C, 69.89; H, 4.54: N, 5.75.

(2) Synthesis of NE-OTf Triple o-methanesulfonic anhydride (5 g, 0.018 mol) was dissolved in dichloromethane (100 ml), and pyridine (1.4 g) was added thereto.
(0.018 mol), 3.9 g of naphthalimide obtained in (1) (0.01 g)
A suspension consisting of 6 mol) and 100 ml of hot dichloromethane was added little by little over 1 hour while maintaining the temperature of the reaction solution at -5 ° C or lower, and then the stirring reaction was continued for 2 hours. After completion of the reaction, the reaction solution was washed three times with cooled deionized water, dehydrated with anhydrous magnesium sulfate, and dried. The solvent was distilled off under reduced pressure,
The obtained residue was recrystallized twice with a mixed solvent of dichloromethane and tetrachloromethane to obtain 3.2 g of NE-OTf transparent flakes. Yield 53%.

mp 138-140 ° C.

Elemental _{analysis: C 15 H 10 NSO 5 F} 3 Calculated (%): C, 48.26; H, 2.68; N, 3.75.

Found (%): C, 48.06; H, 2.68; N, 3.74.

^{IR (KBr) cm -1: 1200,1400} (-O-SO 2 -).

MS: m / z = 374 (MH ^<+> ).

This compound is extremely stable in a crystalline state and has a room temperature (32
C), no change over time was observed even after storage for 6 months in a desiccator under shading.

Example 2. Synthesis of S-(+)-[1-methyl-2- (2,3-naphthalimino)] ethyltrifluoromethanesulfonate (OPNE-OTf) (1) S-(+)-(2,3- Naphthalimino) -2-
Synthesis of propanol In 200 ml of dry toluene, 5.2 g (0.026 mol) of 2,3-naphthalenedicarboxylic anhydride and S-(+)-1-amino-
2.0 g (0.027 mol) of 2-propanol was added, and a dehydration reaction was performed for 2 hours under heating and reflux. After completion of the reaction, the reaction solution was cooled to collect the product, which was washed three times with 50 ml of cold water three times, and then recrystallized with toluene to obtain 5.0 g of the desired naphthalimide transparent needles. 75% yield.

mp165 ° C.

[Α] _D = + 4.9 ° Elemental analysis: C ₁₅ H ₁₃ NO ₃ Calculated (%): C, 70.59; H, 5.11; N, 5.49.

Found (%): C, 70.32; H, 4.95; N, 5.30.

MS: m / z = 255 (M ⁺ ).

(2) Synthesis of OPNE-OTf Triple o-methanesulfonic anhydride (5 g, 0.018 mol) was dissolved in dichloromethane (100 ml), and pyridine (1.4 g) was added thereto.
(0.018 mol), 4.1 g of the naphthalimide obtained in (1) (0.01 g)
A suspension consisting of 6 mol) and 100 ml of hot dichloromethane was added little by little over 1 hour while maintaining the temperature of the reaction solution at -5 ° C or lower, and the stirring reaction was continued for 2 hours. After completion of the reaction, the reaction solution was washed three times with cooled deionized water, dehydrated with anhydrous magnesium sulfate, and dried. The solvent was distilled off under reduced pressure,
The obtained residue was recrystallized twice with dichloromethane to obtain OPNE-O
2.4 g of transparent flakes of Tf were obtained. 35% yield.

mp 135 ° C (decomposition).

[Α] _D = + 18.78 ° Elemental analysis: C ₁₆ H ₁₂ NSO ₅ F ₃ Calculated (%): C, 49.61; H, 3.10; N, 3.62.

Found (%): C, 49.36; H, 3.18; N, 3.54.

^{IR (KBr) cm -1: 1200,1400} (-O-SO 2 -).

MS: m / z: 387 (M ^<+> ).

Example 3. Derivatization HPLC of saturated fatty acids using NE-OTf as a derivatization reagent <Sample> Mixed solution of 6 kinds of aliphatic saturated fatty acids of C _{6 to} C ₁₆ (even number) <Operation procedure> Acetonitrile solution of sample 18-crown-6 in 0.5 ml
0.1 ml of an acetonitrile solution (10 ^-3 M) and about 5 mg of anhydrous potassium fluoride were added and shaken. Then, 0.1 ml of an acetonitrile solution of NE-OTf (10 ^-3 M) was added thereto, and the mixture was shaken at room temperature for 10 minutes. The mixture was stirred. After the completion of the reaction, the mixture is allowed to stand for 30 seconds, and the supernatant 10
Analysis was performed by injecting μl directly into the HPLC.

<HPLC equipment and measurement conditions> Pump: JASCO 880-PU Injector: Leodyne 7125 Detector: JAI 3702-UV detector (set to 259 nm) and Hitachi F
-1000 fluorescence detector (Ex: 259nm, Em: 394nm) Column: Wakosil 5C18-200T (5μm, 4.6mmφ × 15cm, Wako Pure Chemical Industries, Ltd.) Mobile phase: CH ₃ OH-H ₂ O (9: 1) Mobile phase flow rate: 1.0 ml / min. <Results> Results of analyzing a mixed solution of 6 kinds of C ₆ -C ₁₆ (even number) aliphatic saturated fatty acids of C ₆ -C ₁₆ (even number) according to the above operating procedure under the above HPLC conditions (liquid The chromatogram is shown in FIG.

However, in the figure, 1 indicates the peak of n-caproic acid, 2 indicates the peak of n-caprylic acid, 3 indicates the peak of n-capric acid, 4 indicates the peak of lauric acid, 5 indicates the peak of myristic acid, and 6 indicates the peak of palmitic acid.

As is clear from FIG. 1, the derivatization HPLC using the derivatization reagent according to the present invention provides a liquid chromatogram from which saturated fatty acids have been separated extremely well. The detection limit (S / N = 3) of this reagent was 100 fmol for UV detection and 4 fmol for fluorescence detection.

Example 4. Derivatization HPLC of myristic acid using NE-OTf as a derivatization reagent (preparation of calibration curve) Example 3 using myristic acid at various concentrations as a sample
Each sample was derivatized by the same operation procedure as described above, and HPLC analysis was performed using the same apparatus and the same measurement conditions as in Example 3 (using a separately synthesized lauric acid derivative as an internal standard substance), and the calibration curve was obtained. Created. The results are shown in FIG.

As is clear from FIG. 2, the calibration curve (correlation coefficient r = 0.
9996) was a straight line passing through the origin, indicating good quantitative properties.

Example 5 Derivatization HPLC of fatty acids present in mouse cerebrum using NE-OTf as a derivatization reagent <Sample> A mouse cerebrum was taken out, 0.1 ml of margaric acid was added as an internal standard, and 3 ml of methanol was added. After homogenization, 10
Centrifuged at 5000 g for minutes. The supernatant was collected, the residue was homogenized again with methanol, and centrifuged again to collect the supernatant. After repeating this extraction operation three times, the supernatants were combined, concentrated under reduced pressure to distill off the solvent, and the residue was treated with acetonitrile.
The sample was dissolved in 0.2 ml.

<Operation procedure> Derivatization was performed according to the operation procedure of Example 3, and HPLC analysis was performed in the same manner as in Example 3.

<HPLC device and measurement conditions> Pump: Same as in Example 3.

Injector: Same as above Detector: Same as above Column: Chemcosorb 5C8 (5 μm, 4.6 mmφ × 15 cm, trade name of Chemco) Mobile phase: CH ₃ OH-H ₂ O (87:13) Mobile phase flow rate: 1.0 ml / min <Result> The obtained liquid chromatogram is shown in FIG. However,
In the figure, D indicates the peak of docosahexaenoic acid, A indicates the peak of arachidonic acid, P indicates the peak of oleic acid, M indicates the peak of margaric acid (internal standard), and S indicates the peak of stearic acid.

As is clear from FIG. 3, according to the derivatization HPLC using the derivatization reagent according to the present invention, the actual sample (mouse cerebrum)
Even when is used, a liquid chromatogram in which saturated or unsaturated fatty acids are separated very well can be obtained.

Table 1 shows the measurement results of five kinds of fatty acids contained in 0.33 g (wet) of mouse cerebrum.

As is clear from Table 1, the fatty acids contained in this sample could be measured with a coefficient of variation of about 2%.

In addition, in this derivatization HPLC, mouse | mouth per one analysis
Analysis was possible with a sample of 1/250 animals.

Example 6 Derivatization HPLC of α-methoxyphenylacetic acid using OPNE-OTf as a derivatizing reagent <Sample> RS-α-methoxyphenylacetic acid in acetonitrile <Operation procedure> Derivative according to the operation procedure of Example 3. Then, HPLC analysis was performed in the same manner as in Example 3.

<HPLC device and measurement conditions> Pump: Same as in Example 3.

Injector: Same as above Detector: Same as above Column: Same as above Mobile phase: CH ₃ CN-H ₂ O (6: 4) Mobile phase flow rate: 0.6 ml / min. <Results> The obtained liquid chromatogram is shown in FIG. However,
In the figure, R indicates the peak of the R-(-)-enantiomer of α-methoxyphenylacetic acid, and S indicates the peak of the S-(+)-enantiomer.

As is clear from FIG. 4, the compound of the present invention wherein R is a methyl group in the general formula [I] (having an asymmetric carbon atom)
Can be effectively used as a reagent for separating optical isomers.

〔The invention's effect〕

As described above, the present invention particularly provides a derivatization (labeling) reagent for HPLC which is effective for separation and quantification of a saturated or unsaturated carboxylic acid. , It can be stored at room temperature for 6 months or more, and the derivatization reagent of the present invention can derivatize (label) carboxylic acids at room temperature in a very short time, UV detection, fluorescence detection Both are possible, and in both cases, there is a remarkable effect in that the sensitivity is high, etc., and this is a great invention that contributes to the industry.

[Brief description of the drawings]

FIGS. 1, 3 and 4 show the liquid chromatograms obtained in Examples 3, 5 and 6, respectively.
The figure shows the calibration curve of myristic acid obtained in Example 4 [horizontal axis:
Concentration of myristic acid (× 10 ^-8 M), vertical axis; peak area]
Is shown.

Continuation of the front page (51) Int.Cl. ⁶ identification code FI G01N 30/88 G01N 30/88 C (58) Investigation field (Int.Cl. ⁶ , DB name) G01N 31/22 G01N 30/06 G01N 30 / 88 G01N 21/78

Claims (3)

(57) [Claims] 1. A compound of the general formula [I] (Wherein, R ^{1 to} R ⁶ each independently represent a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom, and R ³ and R ⁴ are bonded to each other to form an aromatic ring. R represents a hydrogen atom or a lower alkyl group;
Represents an integer of 0 to 10. A derivatization reagent for high performance liquid chromatography, comprising a compound represented by the formula: (2) A compound represented by the formula: 3. The expression A compound represented by the formula: