JPS614958A - Analysis of amino glycoside antibiotic in blood - Google Patents

Abstract

PURPOSE:To detect an intended component as one peak by pretreating a serum sample to prepare a sample and separating the sample to the component by a high-speed liquid chromatograph then labeling the component by orthophthanol aldehyde. CONSTITUTION:The serum which is the sample and an initial buffer are injected into a carboxylmethyl (CM) cephadic column which contains internally the CM cephadic.gel (gel) 16 and is provided with a gel bed 17 to clean the tamper in the sample. The alkali buffer is then injected therein after cleaning to elute the component adsorbed to the gel 16. The alkali buffer contg. the intended component is injected into the high-speed liquid column of an analyzing instrument and the column is operated to bring orthophthanol aldehyde into reaction with the separated amino glycoside antibiotics in the blood. The high selectivity and high sensitivity are thus obtained and the intended component is detected as one peak.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Field of Application of the Technology The present invention relates to a method for analyzing blood aminoglycoside antibiotics using high performance liquid chromatography.

Conventional technology Analyzes of blood aminoglycoside antibiotics such as cinmycin include the pre-label method, in which 1-fluoro-2,4 dinitrobenzene is mixed into the sample and analyzed using a high-performance liquid chromatography device, and the high-performance liquid chromatography method. A post-label method is used in which the sample is separated using a graph device and then detected using orthophthalaldehyde, but the former method requires complicated pretreatment and has the problem of low sensitivity and low selectivity. In addition, the latter method has the problem that two peaks are detected for cinmycin, making quantitative work cumbersome.

C. Purpose In view of these problems, the present invention aims to analyze a novel aminoglycoside antibiotic in blood that has high selectivity and sensitivity through simple pretreatment and can detect the target component as a single peak. The purpose is to propose a method.

22 The constitution of the invention, that is, the characteristics of the present invention, is that a serum sample is pretreated with a carpogycyl methyl Sephadex column to obtain a sample, and after separation into components by high performance liquid chromatography, orunphthanol The point is that it is labeled with an aldehyde.

E. Embodiments The present invention will now be described in detail based on illustrated embodiments.

FIG. 1 shows an example of an apparatus used in the present invention, in which a high-performance liquid column 1, a reaction coil 2, and a fluorescence detector 3 are connected in series, and a pump is connected to the connection point between the column 1 and the reaction coil 2. The ormphthalaldehyde reaction reagent is supplied from the reagent tank 5 via the reagent tank 4. In the figure, reference numeral 6 denotes a column constant temperature bath that maintains the column 1 and reaction coil 2 at a constant temperature, and 7 denotes a mobile phase liquid feeder that feeds the carrier liquid from the carrier liquid tank 9 to the column via the injector 8. 10 indicates a pump, and IO indicates a recorder connected to a fluorescence detector 3, respectively.

Figure 2 shows a carboxymethyl cephatainic acid ram (hereinafter referred to as C
This is an example of the M-Sephadex power ram), which has an injection port 1 that can be tightly closed with a cap 11 on the top.
2, and a discharge port 14 that can be tightly plugged with a camp 13 at the bottom.
The column body 15 is provided with a carboxymethyl methane gel 16 contained therein. Note that the reference numeral 17 in the figure indicates a filter member provided below the gel ped floor.

Next, the operation of the apparatus configured in this way will be explained.

Serum as a sample and initial buffer are injected into the CM Sephadex column to wash away proteins in the sample.

After washing, alkaline buffer is injected to elute the components adsorbed on the CM Sephadex gel. When the alkaline buffer containing the target component is injected into the high performance liquid column 1 (Figure 1) and the analyzer is operated, orthophthanol aldehyde is added to the blood aminoglycoside antibiotics separated by column 1. reacts and is specifically detected by the fluorescence detector 3.

[Example] (a) Sample 0M containing 0.9 mfl of CM Sephadic gel
Add 400 g J1 of sample serum and 0 to the Sephadic column.
．． Wash with 2 m of 4 molar sodium acetate, and after draining the fluid components, wash again with 2 m of 0.4 molar sodium acetate. After draining the fluid components, 600 g of 0.4 M sodium hydroxide (alkaline buffer) to which 10 mmol of sodium hydroxide was added was injected, the column was shaken, and an additional 400 g of alkaline buffer was added to prepare the CM Sephadex gel. A sample is prepared by eluating the target component from the sample.

(b) Mobile phase 25 mmol of sodium paratoluenesulfonate was dissolved in distilled water as an ion pair sample, and 20 mmol of sodium phosphate was added to provide buffering properties, and perchlorine was added to speed up the elution of the target component. acid sodium to 0.
2 to 1.0 mol was added, and the hydrogen ion concentration was adjusted to 2.0 with phosphoric acid.

(C) Column for sirimycin, astromycin, tobramycin and netilmicin, inner diameter 4.6 mm, length 150 m
octylsilane-bonded silica gel with a particle size of 5 Bm is filled in a column container of 1.0 m, and nectadecylsilane-bonded silica gel with a particle size of 5 gm is filled in the same blade ram container for micronomycin analysis.

(d) Reaction coil A stainless steel tube with an inner diameter of 0.5 mfn and a length of 700 mm was formed into a coil shape.

(e) Fluorescence detector has maximum spectral intensity at wavelength 360 nm, wavelength range 3
Equipped with a high-pressure mercury lamp with spectral characteristics from 00 nm to 450 nm as a light emitting part, and an emission filter that transmits wavelengths of 430 nm or more.

(f) Reagent: 9.4 g of formic acid, 4.8 g of sodium hydroxide and 0.5 g of surfactant are dissolved in distilled water, 4'00 mg of orthophthalaldehyde is dissolved in 6 mg of ethanol, and then 2
- 1 mJ2 of methicaptoethanol was mixed, the total amount was adjusted to 500 mM with distilled water, and the pH was adjusted to 10.5 with sodium hydroxide.

After completing these preparations, tobramycin 10p, u at a concentration of 9.4 ml/m was injected into the column as an internal standard solution, and at the same time, the mobile phase was 0.8 ml/min and the reagent was 0.0 ml/min.
When the analysis was carried out while supplying at a flow rate of 5 m/min, peak P1 of tobramycin, which is an internal standard solution, was obtained after about 8 minutes as shown in Figure 3, and no peak appeared after that, indicating that there was no interference. It was confirmed that no peak occurred.

When the 10w sample described above was injected into a high-performance liquid column and analyzed under the same conditions as above, as shown in Figure (B), the peak P1 of tobramycin, which is the internal standard solution, was detected.
After that, sisomicin could be detected as a single peak P2.

Next, replace the column with one for micromycin analysis,
When cinmycin was analyzed as an internal standard solution, the fourth
As shown in Figure (A), peak P3 of cinmycin was obtained after about 3 minutes, and at the same time it was confirmed that no interfering peaks were generated.

When the sample obtained by the above-mentioned treatment was injected into a column and analyzed, a micromycin peak P4 was obtained as shown in Figure (B).

As described above, according to the present invention, a serum sample is pretreated with a carpoxyl methyl Sephadic column to obtain a sample, which is separated into components by high performance liquid chromatography, and then treated with orthophtanol aldehyde. Labeling not only allows serum samples to be pretreated using a simple method, but also allows the detection of the target aminoglycoside antibiotic in blood with high selectivity and sensitivity. Furthermore, the target component can be detected as one peak', thereby simplifying the quantitative work.

[Brief explanation of drawings]

FIG. 1 is a configuration diagram showing an example of a device used in the present invention;
FIG. 2 is a sectional view showing an example of a CM Sephadex force ram used in the present invention, and FIG. 3.4 is a waveform chart showing an example of analysis results according to the present invention.

Claims (1)

[Claims] A step of eluting a sample containing a blood aminoglycoside antibiotic from a serum sample using a carboxymethyl Sephadex column, a step of separating the sample into components by high performance liquid chromatography, and a step of labeling with orthophthalaldehyde. A method for analyzing blood aminoglycoside antibiotics, which comprises the steps of: