JPS63210776A - Selective determination of amino acid - Google Patents

Abstract

PURPOSE:To realize a method for determination of amino acids except alpha-amino acid having the sensitivity and accuracy equiv. to the sensitivity and accuracy of the conventional method for determination by adding an isothiocyanic aryl to a sample soln. and extracting away the product of reaction with the alpha-amino acid. CONSTITUTION:Ethanol, etc., are first added to a vital sample and protein is removed by centrifugal sepn. A basic buffer soln. is added to the supernatant thereof, then the isothiocyanic aryl is added thereto and the liquid is stirred to effect reaction. A proper amt. of water is added to the liquid after the reaction and the extraction is executed with a weakly alkaline and water insoluble org. solvent so that the excess reagent, etc., are migrated to the org. solvent layer. The water layer is then converted to acidity by a mineral acid and is heated to effect reaction. This reaction liquid is concd. and dried under a reduced pressure and is converted to weak alkalinity by a boric acid buffer soln., etc. the liquid is then subjected to an extraction operation by the org. solvent to migrate the thiohydantoin deriv. of the alpha-amino acid to the org. solvent. The liquid is thereafter converted to acidity by hydrochloric acid, etc., and is then extracted by the org. solvent. The org. solvent is removed from this extraction liquid and a proper volume thereof is subjected as the soln. of the eluate of liquid chromatography to high-performance liquid chromatography, by which the determination of the amino acids with high accuracy is permitted.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for fractionating and quantifying amino acids other than α-amino acids in biological samples.

<Prior Art> Conventionally, various amino acids have been administered to living bodies as foods and medicines, and the method of fractionating and quantifying these externally administered amino acids has become an important problem.

Especially for amino acids used as medicines, α-
Many amino acids other than amino acids are used, and in living organisms they coexist with α-amino acids, which constitute most of food and biological components. Various methods for quantifying amino acids other than α-amino acids in various organs of living organisms have been studied.

Taking tranexamic acid (ω-amino acid), which is well-known as a pharmaceutical, as an example, the bioassay method (l
ysis of clot method), chemical analysis method, electrophoresis method, paper chromatography method, ion exchange column chromatograph method, gas chromatography method, gas chromatography-mass spectrometer method, amino acid analyzer method, fluorescamine derivative or ortho Liquid chromatography methods using fluorescence detection of phthalaldehyde derivatives have been developed.

However, bioassay methods have drawbacks such as requiring skilled techniques and insufficient sensitivity, and the recently announced gas chromatography method, gas chromatograph-mass spectrometer method, amino acid analyzer method, and fluorescence detection method The liquid chromatography method is not necessarily a method that can be generally used with high accuracy because it is difficult to maintain and operate the device and is economically disadvantageous.

<Problems Solved by the Invention> As a result of intensive study by the present inventors on a simple method for analyzing the content of amino acids other than α-amino acids administered as pharmaceuticals in biological samples, we found that We have discovered a quantitative method that can utilize a liquid chromatograph equipped with a UV detector, which is easy to operate, and completed the present invention.

(Structure of the Invention) To carry out the present invention, first, ethanol, a lower alcohol such as methanol, acetone, or acetonitrile, preferably ethanol, is added to a biological sample (e.g., human serum, blood plasma, or urine), and proteins are removed by centrifugation. do. The amount of sample used is usually about 0.5 ml. Add a basic buffer such as borate buffer or a basic organic solvent such as pyridine to the supernatant to maintain the pH at about 7 to 11, and then add phenyl isothiocyanate, Aryl isothiocyanates such as naphthyl isothiocyanate, dansyl isothiocyanate, dabcyl isothiocyanate, fluoreracene isothiocyanate, preferably phenyl isothiocyanate (hereinafter referred to as PITC)
(referred to as ) is added thereto, and the mixture is stirred and reacted at room temperature to 100°C for several minutes to several days. The amount of aryl isothiocyanate used is usually 2
Add 0 reaction regression amount of water, which may be about 0 mg, and extract with a water-insoluble organic solvent such as xylene, benzene, toluene, isopropyl ether, chloroform, ethyl acetate, preferably xylene under weak alkalinity, and remove excess water from the organic solvent layer. Transfers reagents and their decomposition products as well as substances with ultraviolet absorption in biological samples. Next, the aqueous layer is acidified with a mineral acid such as trifluoroacetic acid, acetic acid, hydrochloric acid, etc., and heated at room temperature to 100° C. for several minutes to several hours to react. The reaction solution was concentrated to dryness under reduced pressure. The mixture is made weakly alkaline with an IN aqueous sodium hydroxide solution, a boric acid buffer, etc., and extracted with an organic solvent such as benzene or toluene to transfer the α-amino acid thiohydantoin derivative to the organic solvent. Thereafter, the aqueous layer is acidified with hydrochloric acid or the like, and then extracted with an organic solvent such as benzene, chloroform, or ethyl acetate. By distilling off the organic solvent from the extract and applying it to high performance liquid chromatography as a solution of an appropriate amount of liquid chromatography eluent, the analyte and internal standard substance (hereinafter referred to as 1.S) can be retained on the chromatogram. There are almost no interfering peaks in the portion corresponding to time, and quantification can be performed with extremely high precision.

The eluent and packing material for this liquid chromatography vary depending on the type of amino acid to be analyzed. For example, in the case of tranexamic acid, a mixture of phosphoric acid solution and ethanol (
90:10) as eluent and 0ctyl-based cosmos.
il, C, (manufactured by Gyudon Kagaku Co., Ltd.) can be used as a filler.

The quantitative method varies depending on the type of isothiocyanate ester used; for example, when using PITC, 254
The absorbance at a wavelength of n++, when naphthyl isothiocyanate is used, the absorbance at a wavelength of around 295 nm, and when dansyl isothiocyanate is used, the absorbance at a wavelength of around 300 nm may be measured.

As a result of various studies, r and s are preferably compounds that are structurally most similar to the analyte to be quantified. For example, 1.5. As the binder, 3-aminocyclohexanecarboxylic acid is most preferably used.

Amino acids other than α-amino acids to be subjected to the quantitative method of the present invention include ω-amino acids such as the above-mentioned tranexamic acid,
Examples include γ-aminobutyric acid and various other β-amino acids. The content in these samples is extremely small (for example, tranexamic acid in serum is 0.05
μs/mβ or higher), and is a very practical quantitative method.

In order to confirm the reliability of the above quantitative method, samples that had already been measured using an amino acid analyzer were analyzed using this analytical method. As a result, the correlation coefficient between both analytical methods was 0.997 or more.
The high accuracy of this analytical method was confirmed.

<Effects of the Invention> The method of the present invention does not require particularly skilled techniques, uses commonly used equipment, has simple operations, has sensitivity and accuracy comparable to conventional quantitative methods, and is readily available. The present invention has provided a method for quantifying amino acids other than α-amino acids, which allows the use of easy-to-use internal standard substances and is fully applicable to biological samples.

The present invention will be explained below with reference to Examples.

Example 1 Eight different amounts of tranexamic acid, ranging from 0.022 μg to 11.26 μg, were added to 80.5 ml of normal human serum not taking any drugs to prepare samples for a calibration curve.

Three samples of each concentration were prepared. To these samples, 2.92 μg of 3-aminocyclohexanecarboxylic acid as an internal standard substance was added as a 5 μl aqueous solution and mixed, and then 2 ml of ethanol was added and shaken. It was centrifuged and the supernatant was strained into another test tube. Add 1 ml of 0.01 M borate buffer (pH 9,2) and 13 μl of phenyl isothiocyanate to the supernatant, mix, and react at 40°C for 30 minutes. Add 3 ml of xylene to the 0 reaction solution, shake, and centrifuge. The xylene layer was discarded. This operation was performed three times.

Concentrated hydrochloric acid (Lit) was added to the aqueous layer, and the mixture was reacted at 80° C. for 10 minutes, and then concentrated to dryness under reduced pressure. Add 0.1M borate buffer (p
)19.2) 1 ml was added and dissolved. Benzene 2m
1 was added, shaken, centrifuged, and the benzene layer was discarded twice. 0.3 ml of N-hydrochloric acid was added to the aqueous layer, followed by 2 ml of benzene, followed by shaking and centrifugation, and the upper layer was transferred to another test tube. After distilling off the benzene with a nitrogen stream,
The residue was washed with high performance liquid chromatography (hereinafter referred to as HPLC) eluent [2
0ho M phosphoric acid! ! Solution (PH7,08) - Ethanol (
90:10)]0055m and injected approximately 10 μl into HPLC for analysis.

HPCL uses CosmosilsCa on Hitachi 655 type equipment.
A column (4,8++ m i, d, x 15 cm, Gyudon Kagaku ■) was attached, and the eluent was used as the B mobile phase.
The flow rate was 1/min.

Detection of the chromatogram was performed by absorbance at 254Qm. The tranexamic acid derivative eluted at a retention time of about 10 minutes, and the internal standard derivative eluted at a retention time of about 13 minutes. The height of each peak was measured and the ratio thereof was calculated according to the following formula.

The results are shown in Table 1. Then, a calibration curve was determined from the obtained ratios according to the least quadratic method.

Table-1 Serum tranexamic acid calibration curve 11.28 22.52 5.11G11
．． 26 22.52 5. QOoll, 2
6 22.52 5.1305.63
11.2 [i 2.55G5.63
11.26 2.5305.63 1
1.2 [i 2.5802.82 5.
64 1.2002.82 5.64
1.1702.82 5.64 1.2
001.13 2.26 0.4801.
13 2.26 0.4801.13
2.26 0.43G0.45 0
．． 90 0.1700.45 0.90
0.170Q, 4S O,900,17
00,110,220,044 0,110,220,041 0,110,220,043 ・0.055 0.11 0.0190.
055 0.11 0.0230.055
0.11 0.0210.022
0.044 0.0090.
022 0.044 0゜0110.02
2 0.044 0.009Y=0.45
31X-0,02596 ('r=0.99975)*:
As is clear from the correlation coefficient table above, the calibration curve according to the quantitative method of the present invention showed excellent linearity.

Example 2 0.5 ml of normal human urine not taking drugs
Add 4 different amounts from 6 μg to 11.28 μg, 2 ml instead of 1 ml of 0.1 M borate buffer,
The same treatment as in Example 1 above was performed except that 0.6 ml of N-hydrochloric acid was used instead of 0.3 ml, and the peak height ratio was determined.

The results are shown in Table 2. Then, a calibration curve was determined from the obtained ratios according to the least quadratic method.

Table-2 Calibration curve of urinary tranexamic acid 0.56G 1.12 0.250Y
=0.5020X +0.0430 (r-0,9
9514) As is clear from the above table, the calibration curve obtained by the quantitative method of the present invention showed excellent linearity.

Test Example A serum sample with unknown tranexamic acid concentration was treated in the same manner as in Example 1 above, and the peak height ratio and Table 1
The concentration of tranexamic acid was determined from the calibration curve.

The same samples were quantified using a conventional amino acid analyzer. The correlation between the results obtained by the two methods was investigated, and the results are shown in Table 3.

Table-3 Correlation between quantitative values of amino acid analyzer method and this method: 0.4
24 0.3751.10
8 1.055 3.6811 3.4704.876
4.9904.474
4.633 3.952 4.185 2.176 2.307 1.390 1.144 r = 0.9965 As is clear from the above table, the quantitative method of the present invention shows excellent correlation with the conventional quantitative method. Ta.

Claims (1)

[Claims] Add aryl isothiocyanate to the sample solution and
A method for quantifying amino acids other than α-amino acids, characterized by extracting and removing reaction products with amino acids