JP2726900B2 - Quantitative analysis of carboxylic acids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention relates to a high-performance liquid chromatography
A method for quantitative analysis of nitrophenylhydrazide carboxylic acids, which is capable of detecting carboxylic acids in living bodies such as serum and urine or carboxylic acids in foods such as oils and fats and milk in a short time with high accuracy and high sensitivity. About.

(Prior Art) Various carboxylic acids, such as short-chain or long-chain monocarboxylic acids, polycarboxylic acids, and hydroxycarboxylic acids, are present in living organisms or foods. Conventionally, a gas chromatography method or a high performance liquid chromatography (hereinafter abbreviated as HPLC) method is generally used as a quantitative analysis method for carboxylic acids. Gas chromatography is
This is a method in which carboxylic acids such as fatty acids are converted into volatile derivatives before measurement. On the other hand, the HPLC method is a method in which a carboxylic acid such as a fatty acid is converted into a precolumn derivative and then measured.

(Problems to be Solved by the Invention) In living organisms and foods, the concentration of various carboxylic acids is generally extremely low, and coexists with a wide variety of substances. Therefore, conventional measurement methods have high sensitivity and selectivity. Difficult to analyze and the method still has room for improvement. That is, in the gas chromatography method, when a fatty acid is converted into a volatile derivative for measurement, not only the fatty acid is detected but also a by-product of the derivatization is often generated. In the HPLC method, although there is a report that analysis can be performed effectively by precolumn derivatization, at present, only free long-chain fatty acids in blood were detected as examples. Furthermore, in these measurement methods, it is necessary to extract fatty acids from a sample before derivatization, and it is difficult to perform this extraction operation quantitatively. In addition, ester-type fatty acids are decomposed during the extraction process. There are reports that the fatty acids are converted to free fatty acids, which is questionable as a quantitative analysis method.

In some cases, the present inventors have repeated studies and presentations with other researchers in order to solve these problems, and have already directly substituted short-chain and long-chain fatty acids (14 types) in serum with 2-nitrophenylhydrazide derivatives. And quantitatively analyzed by HPLC [Journal of Chromatography 321 , 165
(1985), ibid 333, 215 (1985), ibid 351, 275 (198
6), Clinica Chimica Acta 155, 95 (1986), Journal
of Chromatography 421 , 33 (1987), 416 , 237 (198
7)]. However, there are cases where satisfactory results cannot be obtained with this measurement method, and the main problems can be summarized as follows. First, most of the free fatty acids in serum form a complex with albumin and dissolve, requiring a separate deproteinizing agent to derivatize these fatty acids, During the reaction, the pH of the reaction solution must be adjusted because the pH of the reaction solution is slightly increased by the basic substance in the serum. Second, pH must be adjusted when measuring carboxylic acids in urine. Third, saponification of the sample is necessary first to measure the total fatty acids in fats and oils and milk, and in order to derivatize the fatty acids without extraction after saponification, the saponified sample must be demineralized with a mineral such as hydrochloric acid. Must be neutralized with acid. On the other hand, in this measurement method, since 2-nitrophenylhydrazine hydrochloride is used by dissolving it in a mixed solution of hydrochloric acid and ethanol, the concentration and ratio of hydrochloric acid differ for each analysis sample, and the reagent solution is no longer compatible. 1-ethyl-3- (3-dimethylaminopropyl) used with the above derivatizing reagent
An aqueous solution of carbodiimide hydrochloride may be partially precipitated or decomposed when stored for a long period of one month or more, and there is also a problem in the stability of this derivatization adjusting reagent.

In addition, the analysis by HPLC using another precolumn derivatization reagent can be performed using a reversed-phase C ₈ or C ₁₈ column (a packing material in which octyl or octadecyl groups are chemically bonded to the silica gel surface). _{10: 0 to} C _{22: 6} (where C _{m: n} of m
Is the number of carbon atoms, and n is the number of double bonds due to unsaturated carbon.) Separation of saturated or unsaturated long-chain fatty acids requires 60 minutes or more even when using gradient elution, and the analysis time is considerable. Would be longer. Furthermore, pre-column derivatives such as C ₂ -C ₈ saturated or unsaturated branches, linear fatty acids, C ₃ -C ₈ branches, linear dicarboxylic acids, C ₂ -C ₆ branches, linear hydroxycarboxylic acids, etc. No analysis examples by the HPLC method have been published to date except for the above-mentioned literature.

The present invention has been proposed to solve the above problems, and provides a quantitative analysis method for rapidly and accurately detecting carboxylic acids using 2-NPH.HCl as a precolumn labeling agent. It is an object.

(Means for Solving the Problems) In order to achieve the above object, in the method for quantitative analysis of carboxylic acids according to the present invention, an alcohol solution of 2-nitrophenylhydrazine hydrochloride (hereinafter abbreviated as 2-NPH.HCl) is used. And an analysis sample was dissolved in an alcohol solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as 1-EDC.HCl), and the carboxylic acid was dissolved in 2-ethanol.
After nitrophenylhydrazide, detection is performed by high performance liquid chromatography. As used herein, the term “carboxylic acids” includes saturated or unsaturated, branched and straight-chain monocarboxylic acids, dicarboxylic acids, polycarboxylic acids, and hydroxycarboxylic acids.

In the method of the present invention, carboxylic acids can be directly converted into 2-nitrophenylhydrazide without performing an extraction operation of an analysis sample or the like in advance. 2-NPH ・ H which is a derivatizing reagent
As the alcohol that dissolves Cl, ethanol is preferred among ethanol and methanol, and 1-EDC · H
As the alcohol for dissolving Cl, a pyridine-ethanol mixed solution is preferable. The pH of the analysis sample and the reaction solution is adjusted by adding an aqueous hydrochloric acid solution. The derivatization reaction of carboxylic acids is performed using alcohol, preferably ethanol, which is a solvent for the derivatization reagent, and the ethanol acts as a protein deproteinizing agent for the protein in the analysis sample. In addition, a potassium hydroxide solution, which is one of the derivatization adjusting reagents, has a
It is preferable to change the amount from 10% to 10% and add twice the amount.

In the method of the present invention, typical reagents used for direct derivatization of carboxylic acids are, for example, as follows.

0.02M 2-nitrophenylhydrazine hydrochloride in ethanol (hereinafter abbreviated as 2-NPH.HCl solution) 0.25M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in ethanol (containing 3% pyridine (Hereinafter abbreviated as 1-EDC / HCl solution) 0.2N hydrochloric acid aqueous solution containing 10% (W / V) potassium hydroxide and methanol
1: 4 (V / V) mixed solution 1/30 M phosphate buffer (pH 6.4) and 0.5 M hydrochloric acid 7: 1 (V / V)
In the method of the present invention, 2-nitrophenylhydrazidated carboxylic acids in an analysis sample are preferably detected by an HPLC method, and a reversed-phase column is preferably used in order to obtain better separation results. With a reversed phase column, a C ₁ to C ₁₈ alkyl group is chemically bonded to the silica gel surface by a known method,
The alkyl group generally has C ₁ , C ₄ , C ₈ and C ₁₈ . For example, in the case of C ₁ is bonded trimethylchlorosilane (TMS) silica gel, in the case of C ₄ to bind the n- butyldimethylchlorosilane and n- butyl trichlorosilane silica gel, silica gel n- octyl For C ₈ Dimethylchlorosilane or n-octyltrichlorosilane is bonded. In the case of _C18 , n-octadecyldimethylchlorosilane or n-octadecyltrichlorosilane is bonded to silica gel. The reversed phase column used in the method of the present invention, preferred especially filling the column with filler C _8.
Silica gel used as a carrier for this filler usually has a particle size
10μ or less, average pore size is 60 ~ 300mm, especially particle size 3 ~
It is preferable that the average pore diameter is about 7 μm and the average pore diameter is about 100 °. Detection of dicarboxylic acid or a mixture of mono- and polycarboxylic acids
It may be carried out by reversed-phase ion pair chromatography,
This may be used C ₁₈ column in place of the C ₈ column when.

(Action) In the method of the present invention, 2-nitrophenylhydrazine derivatization can be carried out with half the amount of a reagent solution as compared with the conventional method by dissolving 2-NPH.HCl and 1-EDC.HC1 in alcohol, and And the pH of the reaction mixture can be easily adjusted by adding hydrochloric acid, for example. Alcohol solutions of 2-NPH.HCl and 1-EDC.HCl are common to almost all analysis samples, and can be stored for a longer period (several months) than before. In addition, the potassium hydroxide solution, which is one of the derivatization adjusting reagents, can be changed from 15% in which precipitation and the like are not good for long-term storage to 10% in which these problems do not occur. Ethanol concentration in the reaction mixture is 70-75%
(V / V), and the heating temperature may be about 60 ° C.
By heating the reaction mixture, the carboxylic acids bound to the protein are easily deproteinized to form 2-nitrophenylhydrazine derivatives together with the unbound carboxylic acids. Therefore,
Not only serum fatty acids, but also urine, fats, milk, wine,
It becomes possible to measure carboxylic acids contained in sake, juice, sauce, soy sauce and the like.

The characteristic actions in the method of the present invention are further listed as follows.

2-Nitrophenylhydrazide of carboxylic acids is
It is very simple only by sequentially adding each derivatized sample to the analysis sample, and both aqueous and ethanol-based samples can be carried out under mild conditions (eg, about 60 ° C.) in a short time (eg, 20 minutes) in a weakly acidic region. Therefore, during the derivatization reaction,
It hardly causes side reactions such as oxidation of unsaturated carboxylic acids or hydrolysis of ester carboxylic acids.

The coloration of 2-nitrophenylhydrazine derivatives of carboxylic acids is extremely stable, and their maximum absorption wavelength and molar extinction coefficient are related to the type of carboxylic acids (eg, saturated, unsaturated mono, poly, hydroxy carboxylic acids, etc.). , And can be detected with high selectivity and high sensitivity in the ultraviolet or visible region. Further, in the HPLC method, detection using a reversed-phase column (particularly, a C ₈ or C ₁₈ column) is carried out using 2-nitrophenylhydrazine, a labeling agent, which has a small molecular size and high polarity, and thus has almost no difference in molecular structure. Acids can be separated and can be detected in a short time.

By the method of the present invention, the separation of 15 kinds of saturated and unsaturated long-chain fatty acids from C _{10: 0 to} C _{22: 6} is completed within 15 minutes by isocratic elution. Also, C _{2: 0 to} C _{6: 0}
Up to 14 types of saturated, unsaturated, branched, linear short-chain fatty acids,
12 branched, linear dicarboxylic acids from C _{3 to} C ₈ , C _{2: 0}
10 saturated short-chain fatty acids from C _{10: 0 to} C _{8: 0} , C _{10: 0 to} C _{22: 6}
18 kinds of saturation up, long chain unsaturated fatty acids, and 10 kinds of branch to C ₂ -C _6, the separation of the hydroxycarboxylic acid straight chain, completed within 30 min by isocratic elution.

(Examples) Next, the present invention will be described based on examples, but the scope of the present invention is not limited to the examples.

Example 1 Quantitative analysis of long-chain fatty acids in serum Linoleic acid, linoleic acid, dihomo-
14 kinds of long-chain fatty acids (C) including polyunsaturated fatty acids such as γ-linolenic acid, arachidonic acid and eicosapentaenoic acid
_{10: 0 ~C 22:} about _6), the simultaneous analysis by HPLC (reverse-phase C ₈ column) method as follows.

25 μL of serum was added with 10 μL of ethanol containing 2 nmol of margaric acid as an internal standard, followed by 25 mL of 0.2 N hydrochloric acid aqueous solution.
μ, 0.02M 2-NPH ・ HCl solution 100μ and 0.25M 1-EDC
・ Add 100μ of HCl solution sequentially. The mixture is kept at 60 ° C for 20
After standing for 10 minutes, 100 μl of a 10% (W / V) potassium hydroxide solution dissolved in water-methanol (1: 4, V / V) was added.
Leave at 60 ° C for 15 minutes. The reaction mixture is neutralized by adding 2 ml of a 1/30 M phosphate buffer (pH 6.4) -0.5 M hydrochloric acid (7: 1, V / V) solution, and then extracted with 2 ml of hexane. After distilling off the hexane in a nitrogen stream at room temperature from hexane layer, the residue was dissolved in methanol 50.mu., a single mobile phase by reversed phase C ₈ column and the 2～10Myu (acetonitrile - water)
, A simultaneous analysis of 15 long-chain fatty acids is achieved within 15 minutes. The result is shown in FIG.

Using this method, quantitative analysis of free long-chain fatty acids in serum in healthy subjects and type II diabetics shows that serum 25
The measurement results under the above-mentioned HPLC analysis conditions using μ are as shown in FIGS. 2 (1) and (2). From FIGS. 2 (1) and (2), it can be seen that the average value of the total concentration of free long fatty acids in the serum of the diabetic patient is about 2.5 times higher than that of the healthy subject. Furthermore, it can be seen that in diabetic patients, the compositions of stearic acid and arachidonic acid are significantly reduced, while linolenic acid is significantly increased.

Example 2 Quantitative Analysis of Short-Chain Fatty Acids in Serum Crotonic acid, β-methylcrotonic acid,
Unsaturated or 13 kinds of short-chain fatty acids containing a branched fatty acid such as tiglic acid for _{(C 2 ~C 6), HPLC} ( reverse-phase C ₈ column)
Simultaneous analysis by the method is performed as follows.

100 nm of serum, 1 nmol of 2-ethylbutyric acid as internal standard
Was added, followed by 100 μL of a 0.2 N hydrochloric acid aqueous solution, 200 μM of a 0.02 M 2-NPH.HCl solution and 0.25 M 1
-Add 200 μl of EDC / HCl solution sequentially. Mix 60
After leaving at 20 ° C. for 20 minutes, 200 μm of a 10% (W / V) potassium hydroxide solution dissolved in water-methanol (1: 4, V / V) is added, and further left at 60 ° C. for 15 minutes. 1/30 to this reaction mixture
4 ml of M phosphate buffer (pH 6.4) -0.5 M hydrochloric acid (7: 1, V / V) solution
And neutralized, and washed twice with 4 ml of hexane. Take 3 ml of the aqueous layer, extract with 4 ml of ether, and wash the ether layer with 4 m of water.
After washing with water, the ether is distilled off at room temperature in a stream of nitrogen. The residue was dissolved in 50 μl of methanol.
The 10μ single mobile phase by reversed phase C ₈ column - detects by (acetonitrile-water), to achieve the simultaneous analysis of 14 types of short-chain fatty acids within 24 minutes. The result is shown in FIG.

When this method is used to quantitatively analyze the free short-chain fatty acids in the serum of healthy subjects and those with abnormal branched-chain amino acid metabolism, the measurement results under the above-mentioned HPLC analysis conditions using 100 μ of serum are shown in FIG. And (2).

Example 3 Quantitative analysis of hydroxycarboxylic acid in urine 1 ml of ethanol containing 200 nmol of an internal standard in 200 μ of urine
, Followed by 100 μM of a 0.2N hydrochloric acid aqueous solution, and the solvent is distilled off in a nitrogen stream at room temperature. 100 μL of a 0.2N hydrochloric acid aqueous solution was added to the residue, followed by 200 μL of water, 0.02 M 2-NPH
200 μl of HCl solution and 200 μl of 0.25 M 1-EDC.HCl solution are added sequentially. After leaving this mixture at 60 ° C. for 20 minutes,
200 μm of a 10% (W / V) potassium hydroxide solution dissolved in methanol (1: 4, V / V) is added, and the mixture is further left at 60 ° C. for 15 minutes. This reaction mixture was added to a 1/30 M phosphate buffer (pH 6.4) -0.1.
After adding 4 ml of a 5M hydrochloric acid (7: 1, V / V) solution, the mixture is washed twice with 4 ml of hexane. An aqueous layer (3 ml) is taken, extracted twice with ether (4 ml), and the ether layer is dried over sodium sulfate, followed by distilling off ether in a nitrogen stream at room temperature. Residue in methanol 50μ
And 10 to 10 μl thereof are analyzed simultaneously by HPLC (reverse phase C ₈ column) to detect 10 types of hydroxycarboxylic acids.

Example 4 Quantitative analysis of dicarboxylic acid in urine 2 ml of ethanol containing 100 nmol of an internal standard in 500 μ of urine
Is added, followed by 200 μm of a 0.2N hydrochloric acid aqueous solution, and the solvent is distilled off at room temperature in a nitrogen stream. 100 μL of a 0.2N hydrochloric acid aqueous solution was added to the residue, followed by 200 μL of water, 0.02 M 2-NPH
200 μl of HCl solution and 200 μl of 0.25 M 1-EDC.HCl solution are added sequentially. After leaving this mixture at 60 ° C. for 20 minutes,
200 μm of a 10% (W / V) potassium hydroxide solution dissolved in methanol (1: 4, V / V) is added, and the mixture is further left at 60 ° C. for 15 minutes. This reaction mixture was added to a 1/30 M phosphate buffer (pH 6.4) -0.1.
After adding 4 ml of a 5M hydrochloric acid (7: 1, V / V) solution, the mixture is washed twice with 4 ml of ether. Take 3 ml of aqueous layer, and add 0.2N hydrochloric acid solution 100μ
Was added thereto, and the mixture was extracted twice with 4 ml of ether. The ether layer was dried over sodium sulfate, and the ether was distilled off at room temperature in a nitrogen stream. The residue was dissolved in 50 μl of methanol,
Simultaneous analysis of 1010 μm by HPLC (reverse-phase _C18 column) method detects 12 kinds of dicarboxylic acids.

Example 5 Quantitative analysis of long-chain fatty acids in fats and oils or milk Take 0.1 ml of a solution prepared by dissolving 1 mg of fats and oils in 1 ml of chloroform, distill off chlorophorom in a nitrogen stream at room temperature, and use the obtained residue as an analytical sample. .

To this analysis sample, add 100 μl of a 2N potassium hydroxide-ethanol (1: 4, V / V) mixture containing 50 nmol of an internal standard, and add
To complete the saponification. To this was added 200 μL of a 0.2N hydrochloric acid aqueous solution, followed by 20 mL of a 0.02 M 2-NPH.HCl solution.
0 μ and 200 μ of 0.25 M 1-EDC.HCl solution are added sequentially. After leaving this mixture at 60 ° C. for 20 minutes, 200 μm of a 10% (W / V) potassium hydroxide solution dissolved in water-methanol (1: 4, V / V) is added, and further left at 60 ° C. for 15 minutes. I do. After adding 4 ml of a 1/30 M phosphate buffer (pH 6.4) -0.5 M hydrochloric acid (7: 1, V / V) solution to the reaction mixture, the mixture is extracted with 4 ml of hexane. After distilling off the hexane from the hexane layer at room temperature in a nitrogen stream, the residue was dissolved in 50 µ of methanol, and 2 to 10 µ of the residue were analyzed simultaneously by HPLC (reverse phase C ₈ column). Is detected.

In the case of milk, 10 μ is used as an analysis sample, and the analysis sample is treated in the same manner as described above to detect 18 kinds of long-chain fatty acids.

Example 6 Quantitative Analysis of Short-Chain Fatty Acid in Fat or Oil or Milk 1 mg of fat or oil was treated in the same manner as in Example 5 to form 2-nitrophenylhydrazide, and the obtained reaction solution was added to a 1/30 M phosphate buffer (pH 6. 4) After adding 4 ml of -0.5 M hydrochloric acid (7: 1, V / V) solution, wash with 4 ml of hexane. Take 3 ml of aqueous layer, extract with 4 ml of ethyl acetate, wash the ethyl acetate layer with 4 ml of water,
Ethyl acetate is distilled off at room temperature in a nitrogen stream. The residue was dissolved in methanol 50.mu., when the 5~10μ analyzed simultaneously by HPLC (reversed phase C ₈ column) method, to detect the 10 kinds of short-chain fatty acids.

In the case of milk, 10 μ is used as an analysis sample, and the analysis sample is treated in the same manner as described above to detect 10 types of short-chain fatty acids.

(Effect of the Invention) The quantitative analysis of carboxylic acids in living bodies such as serum and urine or foods such as fats and oils and milk by the method of the present invention can be carried out by a simple operation, and can be performed in a short time with high accuracy and high sensitivity. Carboxylic acids can be detected. Since various carboxylic acids in blood, urine, or tissue mucosa have different appearances in a normal state and a disease state, if these carboxylic acids are accurately and rapidly detected, biochemical research and diagnosis of disease, Furthermore, it can provide important information on the management of the patient's condition. Also,
Detection of carboxylic acids in fats and oils and milk by the method of the present invention can provide important information on quality checks and product management. Therefore, the present invention can be widely applied not only as a simple quantitative analysis method for carboxylic acids but also in pathological biochemical research or clinical aspects, or in the field of food industry.

[Brief description of the drawings]

FIG. 1 is a chromatogram of 2-nitrophenylhydrazine derivatives of 15 kinds of long-chain fatty acids, FIG. 2 (1) is a chromatogram similar to FIG. 1 for serum of a healthy subject, and FIG. 2 (2) is II. FIG. 3 is a chromatogram of 2-nitrophenylhydrazine derivatives of 14 types of short-chain fatty acids, and FIG. 4 (1) is a chromatogram of sera of healthy subjects. FIG. 4 (2) is a chromatogram similar to FIG. 3 relating to serum of a patient with a branched amino acid metabolism disorder.

 ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-63-246659 (JP, A) Ancl. Lett. 15 (A20) P. 1629-1642 Chromatogr, 416 (1987) p. 237-245 Chromatogr, 333 (1985) p. 215-219

Claims (4)

(57) [Claims] An analytical sample is dissolved in an alcohol solution of 2-nitrophenylhydrazine hydrochloride and an alcohol solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, and carboxylic acids are dissolved in 2-nitrophenylhydrazide. Quantitative analysis of carboxylic acids detected by high performance liquid chromatography after conversion. 2. The method according to claim 1, wherein an analysis sample is directly obtained by adding 2-nitrophenylhydrazine hydrochloride to an ethanol solution and 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide hydrochloride dissolved in a pyridine-ethanol solution, adjusted to pH with an aqueous hydrochloric acid solution, deproteinized by heating and 2-nitrophenylhydrazide of carboxylic acids, and then subjected to high-performance liquid chromatography. reverse phase C ₈ or quantitative analysis of the carboxylic acids to be detected by simultaneous analysis using a C ₁₈ column. 3. An ethanol solution of 2-nitrophenylhydrazine hydrochloride which is added as a derivatizing reagent to an analysis sample when a carboxylic acid is converted into 2-nitrophenylhydrazide. 4. A 1-ethyl-3- (3-dimethylaminopropyl) compound to be added as a derivatization adjusting reagent to an analytical sample when a carboxylic acid is converted to 2-nitrophenylhydrazide.
A pyridine-ethanol solution of carbodiimide hydrochloride.