JPH0210380B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for analyzing guanidine derivatives and an apparatus for analyzing the same. In recent years, guanidine derivatives have attracted attention as uremic toxins that cause various uremic symptoms in the human body's circulatory system, digestive system, respiratory system, hematopoietic system, and nervous system. Therefore, the importance of analyzing guanidine derivatives in biological fluids for testing renal function is increasing. Conventionally, the following methods have been used for this analysis. That is, the sample solution is subjected to liquid chromatography using a column packed with a strongly acidic cation exchange resin and having an inner diameter of 6 to 8 mm and a length of about 30 cm to elute each guanidine derivative. Next, this eluent was mixed with 9,10-phenanthrenequinone (PQ) in an alkaline solution in a reaction coil with an inner diameter of 0.7 to 1 mm in the fluorescent reaction section.
After reacting with the compound, it is analyzed by fluorescence analysis. However, this conventional method requires a long analysis time, such as 1 to 2 hours, and is difficult to apply to routine analysis. Furthermore, the reaction reagent PQ is not water-soluble, so it is used after being dissolved in ethanol or dimethylformamide, but when mixed with the eluent, a precipitate often forms, causing analysis to be interrupted. This invention was made in order to solve the above-mentioned drawbacks. A sample containing a trace amount of guanidine derivatives was subjected to high-performance liquid chromatography, and the eluent was successively mixed with an aqueous sodium hydroxide solution and then a ninhydrin aqueous solution. The reaction solution is irradiated with excitation light to cause it to emit fluorescence.
A method for analyzing guanidine derivatives, which is characterized by quantifying the guanidine derivative by measuring its fluorescence intensity; and high-performance liquid chromatography;
A fluorescent reaction section and a fluorescence intensity measuring section for the eluate from the chromatograph are connected in sequence, and the column of the high performance liquid chromatograph has an inner diameter of 5 mm or less, a length of 5 cm or less, and a particle size of about 5 μm. The guanidine is filled with a strongly acidic cation exchange resin, the reaction coil of the fluorescent reaction section has an inner diameter of 0.3 mm or less, and is equipped with an injection means for an aqueous sodium hydroxide solution and an injection means for an aqueous ninhydrin solution in front of the reaction section. The present invention provides a derivative analysis device. The features of this invention are that as a reagent for making guanidine derivatives fluoresce, first an aqueous sodium hydroxide solution is added, and then an aqueous ninhydrin solution, which has not been used in the past, is added, and the column of high performance liquid chromatograph has an inner diameter and a length. is extremely small, the particle size of the strongly acidic cation exchange resin to be filled is small, and the inner diameter of the reaction coil in the fluorescent reaction section is small. Further, according to the present invention, as will be explained in detail below, guanidine derivatives can be quantified in a short time and with high sensitivity. Furthermore, since an aqueous sodium hydroxide solution is added to the eluent and then a ninhydrin solution is added for reaction, ordinary amino acids do not fluoresce. Furthermore, since the fluorogenic reagents sodium hydroxide and ninhydrin are water-soluble, the analysis will not be interrupted due to the generation of precipitating organisms. In addition, with the conventional method, only creatinine, which is present in biological fluids and attracts attention as a uremic toxin, and creatinine, which is the middle of creatine, could be quantified, but according to the present invention, creatine can also be quantified. Furthermore, the separation column is economically advantageous because its inner diameter and length are small.
Easy to put in and take out resin. Therefore, this invention can be said to be particularly excellent as a method for quantifying guanidine derivatives in biological fluids in clinical trials. Samples containing trace amounts of guanidine derivatives in the present invention include biological fluids such as serum, urine, and perfusion fluid. In addition, guanidine derivatives referred to in this invention include guanidine (G), methylguanidine (MG), guanidinoacetic acid (GAA), and guanidinopropionic acid (GPA).
In addition to guanidinobutyric acid (GBA) and guanidinosuccinic acid (GSA), the guanidine-related substances creatine (CR), creatinine (CRN), and arginine (ARG) are also included. In this invention, if the sample is a biological fluid sample, a sample that has been deproteinized is generally used. This sample is first subjected to high performance liquid chromatography. The column used has an inner diameter of 5 mm or less, a length of 50 mm or less, and is filled with a strongly acidic cation exchange resin having a particle size of about 5 μm. An aqueous solution of sodium citrate is used as the mobile phase, and gradient elution is generally performed by sequentially changing the concentration, and the column temperature is about 50°C. Next, first add an aqueous sodium hydroxide solution to the eluent eluted from the high performance liquid chromatograph,
Next, an aqueous ninhydrin solution is added and reacted. Sodium hydroxide aqueous solution is 0.75 to 1.0 normal,
The ninhydrin aqueous solution used has a concentration of 0.4% or more. Next, this mixed solution is sent to the reaction coil (inner diameter 0.3 mm or less) of the fluorescence reaction section, where a fluorescence reaction is carried out at approximately 50°C. It is then sent to a fluorometer and the fluorescence intensity is measured. FIG. 1 shows a flow path diagram of an embodiment of the analyzer of the present invention. In other words, 1 is a stepwise gradient device, 2 is a high-performance liquid chromatography pump, 3 is a sample inlet, 4 is a column, 5 is an aqueous sodium hydroxide solution, 7 is an aqueous ninhydrin solution, and 6 and 8 are reaction reagent pumps. , 9 is a fluorescent reaction section, 10 is a reaction coil, and 11 is a fluorometer. Next, the present invention will be explained in detail using experimental examples and examples. Experimental Example 1 (Study of fluorescence reaction conditions) The results of examining the fluorescence reaction conditions for an aqueous solution of a guanidine derivative will be described. (1) Effect of sodium hydroxide aqueous solution concentration: 1 nmol and 1 nmol each of MG and GSA
A sample of 100 pmol aqueous solution was investigated. In Fig. 1, while column 4 was not filled with anything and a mobile phase of 0.15N sodium citrate aqueous solution was flowing at a flow rate of 0.7ml/min,
The above sample was injected from the sample inlet 3. Next, aqueous sodium hydroxide solutions of various concentrations were added to 0.6
After injecting at a flow rate of 0.6% concentration ninhydrin at a flow rate of 0.4 ml/min,
The reaction was carried out in a reaction coil with an inner diameter of 0.25 mm and a length of 10 m maintained at 50°C, and then the fluorescence intensity was measured to investigate the influence of the concentration of the sodium hydroxide aqueous solution.
The measurement results for MG and GSA are shown in Figures 2 and 3. As a result, it was found that the appropriate concentration of the sodium hydroxide aqueous solution was 0.75 to 1.0 normal. (2) Effect of ninhydrin aqueous solution concentration Samples of 1 nmol and 100 pmol aqueous solutions of MG were studied. In the experiment (1) above, an experiment similar to the above was conducted except that a 0.75N aqueous sodium hydroxide solution and an aqueous ninhydrin solution of various concentrations were used to investigate the influence of the concentration of an aqueous ninhydrin solution. The results are shown in Figure 4, and it can be seen that the concentration of the ninhydrin aqueous solution needs to be 0.4% or more. (3) Effect of fluorescence reaction temperature 1 nmol and 100 pmol aqueous solutions of MG were investigated. An experiment similar to the above experiment (1) was conducted except that the concentration of the aqueous sodium hydroxide solution was set to 0.75 normal and the reaction temperature was changed to examine the influence of the reaction temperature.
The results are shown in Figure 5, and it can be seen that the appropriate reaction temperature is 50°C. Experimental Example 2 (Preparation of calibration curve) Aqueous solutions of GSA and MG at various concentrations were prepared. The fluorescence intensity of each of these sample solutions was measured in the same manner as in Experimental Example 1 under the following conditions. Flow rate of 0.15N sodium citrate aqueous solution:
0.7ml/min Sodium hydroxide aqueous solution concentration and flow rate: 1 normal, 0.6ml/min Ninhydrin aqueous solution concentration and flow rate: 0.6%,
0.6ml/min Fluorescent reaction temperature: 50°C The results are shown in Figure 6, and the coefficient of variation of the measured values was as follows. Concentration Coefficient of variation (%) GSA 1nmol 1.19 〃 100pmol 2.12 MG 1nmol 2.42 〃 100pmol 2.54 From the above results, it was found that approximately 10pmol of the guanidine derivative was detectable, good linearity was obtained down to 1nmol, and good reproducibility was obtained. I know that there is. Experimental example 3 (Study of columns for high performance liquid chromatography) GSA, GAA, GPA, GBA, ARG and MG
An experiment was conducted under the following conditions to investigate the elution behavior of each compound in a mixed aqueous solution with a concentration of 1 nmol. (1) Analytical equipment Mobile phase liquid pump: Shimadzu LC-3A high performance liquid chromatography column: inner diameter 4 mm, length 5 cm, 50°C, coil of fluorescence reaction section filled with strongly acidic cation exchange resin with particle size 5 μm : Teflon inner diameter 0.25
mm, length 10m Fluorometer: Shimadzu RF-500LCA model (Em500nm,
Ex395nm) (2) Fluorescence reaction conditions Sodium hydroxide aqueous solution concentration and flow rate: 0.75 standard,
0.6ml/min Ninhydrin aqueous solution concentration and flow rate: 0.6%, 0.4ml/min
Minute reaction temperature: 50℃ (3) Mobile phase (flow rate 0.7ml/min) 0.2N sodium citrate aqueous solution Elute by changing the pH of the above mobile phase to 3.5, 4.8, 6.0, 11.4, and each volume ratio ( k') was measured and the results are shown in FIG. From the above results, it is clear that according to the present invention, it is sufficient that at most two guanidine derivatives can be eluted in one pH range of the mobile phase. Therefore, it was found that by reducing the particle size of the ion exchange resin packed, it is possible to reduce the inner diameter and length of the column, and as a result, the analysis time can be shortened. Example 1 G, MG, GAA, GPA, GBA, GSA, ARG
and CR concentration is 1 nmol each, CRN concentration is 3μ
g/g of the sample liquid was analyzed using the following analytical equipment under the following analytical conditions. (1) Analytical equipment Stepwise gradient device: Shimadzu SGR
Type 1A mobile phase liquid pump: Shimadzu LC-3A type high performance liquid chromatography column: Internal diameter 4.6 mm, length 3.8 cm, 50°C, strongly acidic cation exchange resin filled with particle size 5 μm Coil of fluorescent reaction section: Teflon inner diameter 0.25
mm, length 10m Fluorometer: Shimadzu RF-500LCA model (Em500nm,
(Ex395nm) (2) Fluorescence reaction conditions Sodium hydroxide aqueous solution concentration and flow rate: 0.75 standard,
0.6ml/min Ninhydrin aqueous solution concentration and flow rate: 0.6%, 0.4ml/min
Min reaction temperature: 50℃ (3) Elution program (mobile phase flow rate 0.7ml/min)

【table】

[Table] The analysis chart obtained is shown in Figure 8, and the measurement was possible in a short time of 32 minutes with good sensitivity.

[Brief explanation of drawings]

FIG. 1 is a flow path diagram of an embodiment of the analyzer of the present invention, and FIGS. 2 and 3 are MG and MG, respectively.
Graphs showing the influence of the sodium hydroxide aqueous solution in the analytical method of this invention for a GSA aqueous solution sample, FIGS. Graph showing the influence of temperature, Figure 6 is a calibration curve for MG and MSG aqueous solutions, Figure 7 is a graph showing the relationship between mobile phase PH and k' (volume ratio) of a trace amount guanidine derivative mixed aqueous solution, Figure 8 The figure is a chromatogram of a trace amount of a mixture of guanidine derivatives and guanidine-related substances obtained by the analytical method of the present invention.

Claims (1)

[Claims] 1. A sample containing a trace amount of guanidine derivatives is treated with a pH value of 3 to 3 using a strongly acidic cation exchange resin.
Each guanidine derivative was separated by high performance liquid chromatography using a gradient elution with a mobile phase changed to 12, and a sodium hydroxide aqueous solution and a ninhydrin aqueous solution were continuously added to the eluent to react. A method for analyzing guanidine derivatives, which comprises irradiating a reaction solution with excitation light to cause it to emit fluorescence, and quantifying each guanidine derivative by measuring the intensity of the fluorescence. 2. A mobile phase gradient supply unit that supplies a mobile phase with a pH value changed from 3 to 12, a high performance liquid chromatograph, a fluorescent reaction unit for eluent from the chromatograph, and a fluorescence intensity measurement unit are connected in sequence. The inner diameter of a high-performance liquid chromatograph column is 5.
mm or less, length is 5cm or less, and particle size is approximately 5μm
The guanidine is filled with a strongly acidic cation exchange resin, the reaction coil of the fluorescent reaction section has an inner diameter of 0.3 mm or less, and is equipped with an injection means for an aqueous sodium hydroxide solution and an injection means for an aqueous ninhydrin solution in front of the reaction section. Analyzer for derivatives.