JPH0240189B2 - KATEKOORURUINOTEISEI * TEIRYOHOHO - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for qualitatively and quantitatively determining catechols. Catechols exist widely in living organisms, and many have important biochemical or clinical chemical meaning. For example, catecholamines, which are derivatives of pyrocatechol, such as 3,4-dihydroxyphenylalanine or 3,4-dihydroxyphenylacetic acid, are closely related to the function of the adrenal medulla, the sympathetic nervous system, and Parkinson's disease. The qualitative and quantitative determination of biosynthetic and metabolic systems has important implications for determining abnormalities in biosynthetic and metabolic systems in living organisms and diagnosing diseases. However, since the amount of these catechols present in living organisms is extremely small, there have been various problems that need to be solved in the qualitative and quantitative methods that have been known or practiced so far. That is, conventional methods for qualitative and quantitative determination of catechols in biological samples include the following, but all of them have drawbacks as described below. (1) Cerium salt method (2) UV method (3) Trihydroxyindole method (4) Ethylenediamine method In the cerium salt method (1) above, a cerium salt aqueous solution is applied to the sample in the presence of ammonia to generate However, this method has the drawback of low detection sensitivity. The UV method described in (2) above uses the fact that catechols have absorption in the ultraviolet region to measure the ultraviolet absorption of the analyte, and has the advantage of being simple to perform. Disadvantages include the absorption of other substances and low sensitivity. The trihydroxyindole method described in (3) above is a method in which the analyte is treated with an oxidizing agent such as Felician ion or iodine to generate hydroxyindole, and its fluorescence is measured. Compared to method (2), highly sensitive analysis is possible, but the operation is complicated, and the sensitivity is extremely low for certain substances such as dopamine and dopa, especially when using chromatography. When used in analysis, it has a serious drawback in that sensitivity equivalent to that of other catecholamines such as epinephrine and norepinephrine cannot be obtained (approximately several tenths to one hundredth). The ethylenediamine method described in (4) above is a method in which ethylenediamine is applied to the analysis sample under alkaline conditions and the fluorescence of the generated fluorescent substance is measured, but because the sensitivity is relatively low, trace amounts of catechol If a more sensitive measurement is desired for analyzing the
It has the disadvantage that satisfactory results are not obtained and therefore its application is limited. The present inventors conducted various studies on qualitative and quantitative methods for catechols, especially for trace amounts of catechols in biological samples. It has been found that by treating with 2-cyanoacetamide and measuring the fluorescence of the resulting fluorescent substance, catechols can be qualitatively and quantitatively determined with excellent results. The present invention is based on this knowledge. The method of the present invention does not have the drawbacks of conventional methods, enables qualitative and quantitative analysis with the same level of high sensitivity for each catechol substance, and has the advantages of simple operation and low cost. It has the excellent advantage of being easy to automate. The present invention will be explained in detail below. In the method of the present invention, an analyte (sample) containing catechols is treated with 2-cyanoacetamide represented by the chemical formula NCCH ₂ CONH ₂ , and the catechols present in the sample are reacted with 2-cyanoacetamide. The catechols in the sample are qualitatively and quantitatively determined by measuring the fluorescence of the fluorescent substance produced by the reaction.
The catechols to be analyzed here have the formula, It is a compound having a catechol structure as shown in Table 1 below, and is a group of substances having a catechol structure, including catecholamines that exist in extremely small amounts as biological components, as shown in Table 1 below. The objects (samples) to be qualitatively and quantitatively measured in the method of the present invention are usually samples of biological origin, such as body fluids such as blood, cerebrospinal fluid, and tissue fluid, and urine, which are to be measured biochemically or clinically. Examples include materials containing catechols. These samples may be subjected to pretreatment, such as removal of impurities or separation of catechols, as necessary.
Subjected to processing to improve qualitative and quantitative accuracy. In order to separate catechols in advance,
Means such as chromatography are used. In the method of the present invention, the analyte as described above is treated with 2-cyanoacetamide,
As a treatment method, any method may be used as long as the catechols in the analyte are brought into contact with 2-cyanoacetamide so that the two react with each other to produce a fluorescent substance. Usually, 2-cyanoacetamide is prepared as an aqueous solution, and this aqueous solution is combined with the object to be analyzed (sample). The concentration of this aqueous solution is usually 0.01w/v
% to 10.0 w/v%, and is preferably used to give a concentration of 0.1 w/v% to 2.0 w/v% in the reaction mixture. When reacting the above-mentioned catechols with 2-cyanoacetamide, more preferable results can be obtained if the reaction is carried out in a buffer solution. The method of the present invention provides more favorable results when carried out in compliance with the following conditions (1) to (6); however,
The implementation conditions are not limited to these. (1) Concentration of 2-cyanoacetamide used It is preferable to use the 2-cyanoacetamide so that the concentration in the reaction mixture is in the range of 0.1 w/v% to 2.0 w/v%.
When the concentration in the reaction mixture is 2.0 w/v% or more, a slight increase in reagent blank is observed. (2) Type and concentration of buffer solution The type of buffer solution is not particularly limited. For example, a phosphate buffer solution, a borate buffer solution, a phosphate-borate buffer solution, etc. are used. The concentration used in the reaction mixture is 0.01M or more (concentration commonly used as a buffer solution). (3) PH of buffer solution: 9 to 13, but the optimum PH varies depending on the individual compound. If the pH deviates significantly from the optimum pH, a decrease in sensitivity can be seen. (4) Reaction temperature Usually 100°C or less is appropriate. At temperatures above 100°C, an increase in fluorescence intensity can be seen, but at the same time consideration must be given to boiling of the reaction solution. (5) Reaction time It can be determined arbitrarily in relation to the reaction temperature, but
At a reaction temperature of about 100°C, the fluorescence intensity reaches its maximum value within a reaction time of about 5 to 10 minutes. (6) Measurement wavelength The maximum excitation wavelength and maximum fluorescence wavelength differ slightly depending on the individual compound (see Table 1), but for qualitative measurements, it is sufficient to irradiate with ultraviolet light and visually measure the fluorescence. . For quantification, the maximum excitation wavelength and maximum fluorescence wavelength of each catechol can be selected and the fluorescence intensity can be measured, or any specific wavelength can be measured by allowing a slight decrease in sensitivity. It is also possible to take the simple method of selectively measuring. In the method of the present invention, qualitative or quantitative determination is performed by measuring the fluorescence of a fluorescent substance produced by the reaction of catechols to be analyzed with 2-cyanoacetamide. When performing this on individual substances separated and eluted by The appropriate method of fluorescence measurement is adopted. The method of quantitative determination by fluorescence measurement is usually a calculation method using a plotted or non-plotted calibration curve, which is generally used in quantitative methods by measuring light intensity.
That is, this is a method in which a calibration curve based on standard concentrations is determined in advance, and the concentration is determined based on the measurement results based on the calibration curve. Examples of the present invention will be described in detail below, including the creation of a calibration curve. Example 1 Solutions of each sample listed in Table 1 (1×10 ^-5 M)
For 1ml, 1ml of 1w/v% 2-cyanoacetamide aqueous solution and 0.3M boric acid buffer solution (PH11.0)
2 ml was added and heated on a boiling water bath for 10 minutes. After cooling, the fluorescence intensity was measured using a spectrofluorophotometer.
Table 1 shows the results.

[Table] Example 2 Epinephrine solution (1×10 ^-8 M to 1×10 ^-4 M)
Add 1 ml of 1 w/v% 2-cyanoacetamide aqueous solution and 2 ml of 0.3 M boric acid buffer solution (PH 12.0) to 1 ml, heat on a boiling water bath for 10 minutes, and after cooling, fluoresce at the wavelength shown in Example 1 and Table 1. The strength was measured. For norepinephrine PH of buffer solution
10.0 and 11.0 for dopamine, and other conditions were the same as those for epinephrine above, and norepinephrine and dopamine were measured to obtain a linear calibration curve. The results are shown in the accompanying drawing, FIG. The vertical axis of FIG. 1 is the relative fluorescence intensity, and the horizontal axis is the concentration (M) of the sample. In Figure 1, E represents epinephrine, NE represents norepinephrine, and DA represents dopamine (hereinafter the same applies in Figures 2 to 5). Example 3 Using the same sample as in Example 2, the sensitivity of the method of the present invention, other conventional methods, trihydroxyindole (THI) method, and ethylenediamine (ED) method was compared for concentrations of 1 to 10 ^-6 M. . The results are shown in Table 2. As can be seen from the second point, the method of the present invention shows superior results in that it has higher sensitivity and less variation in sensitivity depending on the compound than the conventional method.

[Table] Example 4 Hitachi 635 high performance liquid chromatograph with 3011-C
Column filled with gel (inner diameter 2.6 mm, length 25 cm)
was installed to separate and quantify catecholamines at a column temperature of 45°C. 10 μl or 100 μl of sample liquid is
An eluent containing 0.05M dipotassium phosphate in 0.05% phosphoric acid was fed at a rate of 0.60 ml per minute for separation and elution. This eluate was mixed with a 1% 2-cyanoacetamide solution at a rate of 0.050 ml per minute and a buffer solution containing 0.75 M potassium hydroxide in 0.60 M boric acid at a rate of 1.0 ml per minute. Reaction coil (inner diameter
0.5 mm, length 500 cm) and heated to 100°C. Next, this liquid was introduced into a cooling coil and then introduced into a fluorescence detector to measure fluorescence. In addition, if necessary, a chromatogram was automatically recorded and further quantified using an integrator. The time required for the measurement in this example was about 10 minutes. The chromatogram obtained as described above is shown in the attached drawing, FIG. 2. The numerical value is time (minutes), and NE, E, and DA have the same meanings as in Figure 1 (hereinafter, the same applies in Figures 3 to 5). The calibration curve created in this example is shown in the attached drawing, FIG. 3. The vertical axis shows the response value (μV, sec) of the integrator, and the horizontal axis shows the amount of sample (×10 ⁻¹² mol). The attached drawing, FIG. 4, shows a chromatogram obtained by measuring catecholamines in serum using serum as a sample in this example. The attached drawings, FIGS. 5A and 5B, show chromatograms obtained by measuring catecholamines in urine using urine as a sample in this example.
A shows the state at rest, B shows the state after excitement. The analysis results of catecholamines in serum and catecholamines in urine measured in this example are shown below. Results of analysis of catecholamines in serum Epinephrine 0.85ng/ml Norepinephrine 2.6 〃 Dopamine 2.5 〃 Results of analysis of catecholamines in urine At rest Epinephrine 14.1ng/ml Norepinephrine 24.4 〃 Dopamine 254.0 〃 When excited Epinephrine 22.2 〃 Norepinephrine 4 8.9 〃 Dopamine 276.0 〃 Example The amount of sample solution used in 4, the composition and flow rate of the eluent, the concentration and flow rate of the 2-cyanoacetamide solution, the shape of the reaction coil, the reaction temperature, etc. are just examples, and these conditions may vary depending on various requirements. Can be selected arbitrarily. An example of the detection limits in Example 4 is: epinephrine: 0.1×10 ⁻¹² mol, norepinephrine: 0.3×10 ⁻¹² mol, and dopamine: 0.1×10 ⁻¹² mol. In addition, the calibration curve is (1 to 200) ×
10 ^-12 , (1 to 1000) × 10 ^-12 and (1 to 200) ×
It was a straight line in the range of 10 ^-12 moles. In connection with this example, examples of sample pretreatment methods for serum and urine are listed below. Pretreatment of serum 1) Add concentrated hydrochloric acid to commercially available alumina (200 mesh), stir, let stand, and then discard the supernatant. After repeating this operation three times, it is washed repeatedly with distilled water, and when the pH value of the washing solution reaches 4, the alumina is removed and dried at 110°C overnight.
(Alumina pretreatment) 2) Suspend 100 mg of dried alumina in 1 ml of 1M Tris buffer (PH8.7), add 1 ml of blood serum immediately after blood collection, shake for 10 minutes, and then let stand. The supernatant is discarded and washed three times with 5 ml of distilled water and then three times with 3 ml of methanol, and the alumina adsorbed with catecholamines is centrifuged and dried under reduced pressure (adsorption of catecholamines). 3) Add 3 ml of 4M acetic acid to the dried alumina to elute the adsorbed catecholamines, and after centrifugation, concentrate the supernatant to dryness. Dissolve the residue in 90μ of distilled water. (Elution of catecholamines) 4) Inject 10μ of the sample into a separation column and perform chromatography. Pretreatment of urine 1) Add 5.3 ml of 2M hydrochloric acid to 5 ml of urine and heat on a boiling water bath for 20 minutes. (Hydrolysis of catecholamine conjugate) 2) After cooling the mixture to room temperature, 1 ml of 0.05M sodium ethylenediaminetetraacetate solution was added.
Next, adjust the pH to 8.5 with concentrated ammonia water. 3) As in the case of serum, fill 500 mg of pretreated dry alumina into a 1.0φ x 10.0 cm glass tube (with a stopper at the bottom end), add the entire amount of the above solution with the stopper at the bottom end, and shake for 10 minutes. do. 4) Next, open the stopper and let the liquid flow down, then add distilled water.
Wash the alumina using 10ml. 5) Next, flow 5 ml of 0.3M acetic acid to elute the adsorbed catecholamines from the alumina. 6) Inject 10μ of the eluate into the separation column to perform separation. Example 5 (Example of use of thin layer chromatography) Silica gel thin layer plate manufactured by Merck & Co.
Using a 60TLC plate (10 x 20 cm), separately spot 10μ of an aqueous solution containing 0.1 to 10ng of each of epinephrine, norepinephrine, and dopamine, and then mix n-butanol:acetic acid:water (3:1:1). Develop using a solvent. After rolling out, each laminate is air-dried and then
Contains 1.0w/v% 2-cyanoacetamide
Spray 0.3M borate buffer solution (PH: 11.0) evenly. This thin laminate was dried by heating at 110°C for 15 minutes and then allowed to cool. Next, this thin layer plate is set in a densitometer equipped with a mercury lamp that emits strong light at 365 nm, the fluorescence intensity of each spot is measured, and a calibration curve is created for each sample. Under this development condition, the R _f values of epinephrine, norepinephrine, and dopamine are 0.45 and 0.50, respectively.
and 0.55. Next, human urine was treated according to the pretreatment method described above, 10μ of it was developed in the same way, and the spot was identified by emitting light and measuring the R _f value. At the same time, the fluorescence intensity was measured and quantified using the calibration curve obtained above. . The present invention has been described in detail above, and the advantages of the method of the present invention are listed below. (1) It is highly sensitive and allows analysis of extremely small amounts of catechols. (2) There is little variation in sensitivity depending on the type of catechol compounds. (3) All reagents used are inexpensively available and implementation costs are low. (4) Easy to operate. (5) Since the reaction conditions are easy to control and automation is easy, it can be easily incorporated into an automatic analysis system or liquid chromatograph detection system.

[Brief explanation of drawings]

FIG. 1 is a diagram of a calibration curve in Example 1, FIGS. 2, 4, and 5 are chromatograms in Example 4, and FIG. 3 is a diagram of a calibration curve in the same example.

Claims (1)

[Claims] 1. A method for qualitatively or quantitatively determining catechols, which comprises treating a sample to be qualitatively or quantitatively determined with 2-cyanoacetamide and measuring the fluorescence of a fluorescent substance produced thereby.