JP2526574B2 - Assay for tricyclic antidepressants - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring a tricyclic antidepressant drug. More specifically, the present invention relates to a method for measuring a tricyclic antidepressant drug capable of easily and rapidly quantitatively analyzing the concentration of the tricyclic antidepressant drug in a biological sample, particularly blood.

(B) Conventional technology Tricyclic antidepressants such as imipramine and desipramine are
Widely used in the treatment of depression and depressive states, rapid and highly sensitive measurement of these tricyclic antidepressants in blood, especially in blood, has been strongly desired in pharmacokinetics. There is.

However, since various contaminants are mixed in blood, it is not always easy to measure a trace amount of tricyclic antidepressant in the blood.

In this respect, as a conventionally proposed measurement method, there is a method using gas chromatography (detection is FID or FTD).

(C) Problems to be Solved by the Invention However, in the above conventional measuring method using gas chromatography, serum obtained from blood is extracted with n-hexane under alkaline conditions, and the organic phase is extracted with dilute hydrochloric acid. Then, a complicated pretreatment step of extracting the aqueous phase again with n-hexane under alkaline condition, distilling off the organic phase of n-hexane, and dissolving the obtained residue in methanol to prepare a measurement solution. That is, a step of removing impurities was necessary. Therefore, it is difficult to perform rapid measurement, and it cannot be said that the sensitivity is high, and there is a problem in the quantitative accuracy.

The present invention has been made to solve such problems, and in particular, a measurement method capable of rapidly and highly sensitively quantifying a trace amount of a tricyclic antidepressant in a biological sample by a simple pretreatment. Is to provide.

(D) Means for Solving Problems As a result of earnest studies, the present inventor has found that various organic phases obtained only by an extraction treatment with a chlorine-based organic solvent can be obtained without complicated pretreatment. Surprisingly, the fact that tricyclic antidepressants can be detected and quantified with high sensitivity without being affected by contaminating components by applying gas chromatography that detects with a so-called surface ionization detector (SID) among the detection methods And has arrived at this invention.

Thus, according to the present invention, a biological sample is subjected to an extraction treatment with a chlorine-based organic solvent, then the extracted organic phase is directly subjected to gas chromatography, and the fraction thereof is detected by a surface ionization detector to detect a fraction in the biological sample. Provided is a method for measuring a tricyclic antidepressant, which comprises detecting and quantifying a tricyclic antidepressant.

The tricyclic antidepressant which is the subject of the present invention means a dibenzazepine derivative or a similar compound thereof, and representative examples are imipramine, desipramine, clomipramine, aminotriptyline and the like. Further, examples of the sample to be measured include blood, blood supplement, serum, urine, lymph, and the like, and it is particularly preferable to use serum.

The extraction treatment is carried out with a chlorine-based organic solvent, and examples of this solvent include dichloromethane, dichloroethane, chloroform, carbon tetrachloride and a mixed solvent thereof. It is usually suitable to carry out such an extraction treatment under alkaline conditions, and it is preferable to carry out the extraction treatment under conditions of pH 8 to 10 with the addition of a small amount of caustic alkali. By this extraction treatment, the tricyclic antidepressant drug that may be present in the biological sample is extracted into the organic phase together with other extractable impurities.

A part of the organic phase thus obtained is subjected to gas chromatography which is detected by a surface ionization detector (SID). Here, the surface ionization detector (SID) is of a type in which a sample gas is made incident on a high-temperature metal surface in the state of containing no hydrogen gas to emit cations. The detector comprises a heating electrode, a collector electrode for trapping cations or electrons emitted when ionized at the electrode surface, an electrometer for measuring the trapped cations or electrons as an ion current or electron current, and a nozzle electrode. The nozzle electrode is attached to the tip of the sample gas outlet of the separation column, the heating electrode is provided so as to face the nozzle electrode, and the cylindrical collector electrode is arranged so as to surround the electrode. In the method of the present invention, the nozzle electrode is connected to the negative side of the high-voltage power source, a negative DC voltage is applied, and the collector electrode is set to the positive side. Therefore, the flow of electrons when positive ions are generated in the collector electrode is set. It is preferably configured so that it can be measured.

The heating electrode has a large work function and is made of a metal and / or an oxide thereof which is safe under a high temperature of 400 to 700 ° C. Platinum, iridium, molybdenum or rhenium or an oxide of these metals or a mixture thereof. Is suitable.

In the present invention, in order to convey the sample gas, nitrogen,
Although an inert gas such as helium or argon is used as a carrier gas, it is preferable to allow oxygen gas or air to coexist with the gas at least before the detector from the viewpoint of improving sensitivity. In any case, it is necessary to use a carrier gas that does not contain hydrogen.

As a column used for gas chromatography, it is preferable to use a so-called fused silica capillary column which itself has a high resolution in view of the object of the present invention.

(E) Action The organic phase contains many contaminants as well as tricyclic antidepressants, but these contaminants are adversely affected by chromatographic separation and detection by a surface ionization detector. Without separation, each tricyclic antidepressant drug will be clearly separated and detected.

In addition, it is impossible to separate and quantify tricyclic antidepressants due to contaminating components even if the same FID or FTD is used as a detector and subjected to gas chromatography in the same manner.

(F) Embodiment FIG. 1 is a structural explanatory view of an embodiment of a surface ionization detector used for carrying out the method of the present invention. In the figure, a surface ionization detector (hereinafter referred to as SID) 1 is composed of a tubular body having a gas inlet 2 and a gas outlet 3 at both ends. The gas inlet 2
Is composed of a quartz nozzle 5 connected to a separation column 4 of a gas chromatograph (not shown), the open end of the nozzle 5 is open to the ionization chamber 6, and a ring-shaped ring is formed around the open end. Nozzle electrode 7 (made of stainless steel) is provided, and the nozzle electrode 7 is connected to the negative side (-200V) of the external DC power supply 8.

The ionization chamber 6 is provided with an air introduction pipe 9 for introducing oxygen or air near the periphery of the quartz nozzle 5.

In the ionization chamber 6, facing the nozzle electrode 7 at the tip of the quartz nozzle 5, a coil-shaped heating electrode 10 made of a platinum fine wire, a cylindrical inner collector electrode 11 (made of stainless steel) and a cylindrical shape. Outer collector electrode 12
(Made of stainless steel) and the surface ionization detector. The inner collector electrode 11 and the outer collector electrode
The outer collector electrode 12 and the outer collector electrode 12 are formed integrally with each other through an electrically insulating cylinder a made of ceramics. The outer collector electrode 12 projects from the end of the inner collector electrode 11 and surrounds the periphery of the heating electrode 10.

The heating electrode 10 (200V) has one end connected to the end of the inner collector electrode 11 and the other end connected to the end of the outer collector electrode 12, and further connected to the power controller 14 via the transformer 13. , Can be heated to any temperature.

One of the input terminals of the electrometer 15 is connected to one end of the secondary side of the transformer 13, that is, the inner collector electrode 11,
The other is connected to the DC power supply 8. Note that the electrometer 15 is connected to a recorder and a data processing device (not shown).

The surface ionization detector set as described above is configured so that the electron meter 15 detects the electron flow emitted when positive ions are generated in the heating electrode.

A method for carrying out the present invention will be described by gas chromatography using the above detector.

First, 50 μl of serum containing three types of tricyclic antidepressants
Was collected in Micro Spitz, and dichloromethane 50
0 μl was added and 300 μl of 4N potassium hydroxide was further added. The mixture was vigorously shaken in a thermomixer for 1 minute and then centrifuged at 12000 rpm for 10 minutes to sufficiently separate an aqueous phase and an organic phase. After such extraction treatment, 1 μl of the extracted organic phase was sampled and subjected to gas chromatography using SID under the following conditions.

Column: Chemical bond type fused silica capillary column CBP1-S50-050 (Nonpolar 0V-1 equivalent) 50m × 0.33mm Column temperature: 270 ℃ Carrier gas: Helium 4.5ml / min Air flow rate: 150ml / min Drug concentration in serum: The chromatogram thus obtained is shown in FIG. In the figure, is imipramine, is desipramine,
Indicates clomipramine. In this way, all tricyclic antidepressants have a highly sensitive peak and are separated from other contaminants as well as each antidepressant, and quantification is based on the integrated intensity and peak height of this peak. You can see that it can be done easily.

As a result of evaluation using a standard substance, according to the method of the present invention, it was found that each antidepressant drug can be detected to the picogram order or less, and that the calibration curve has linearity within the range of 1 to 100 ng / ml.

It was also found that the analysis time required for one measurement is less than 20 minutes, which can be remarkably shortened as compared with the conventional method.

(G) Effect of the Invention According to the method of the present invention, (1) extraction can be performed only once, so measurement can be performed quickly and easily. (2) tricyclic antidepressant is selectively and highly effective Sensitivity can be detected. (3) Since complicated pretreatment is not required, the sample state is measured in a state close to the raw sample, the probability of error is reduced, and highly reliable data can be obtained. Is expressed.

Therefore, it enables rapid and highly sensitive analysis of a blood level of a tricyclic antidepressant, and its utility is extremely high especially in the field of clinical analysis.

[Brief description of drawings]

FIG. 1 is a configuration explanatory view showing an embodiment of a surface ionization detector used in the measuring method of the present invention, and FIG. 2 is a graph diagram illustrating a chromatogram obtained by the measuring method of the present invention.

Claims (2)

(57) [Claims] 1. A biological sample is subjected to an extraction treatment with a chlorine-based organic solvent, the extracted organic phase is directly subjected to gas chromatography, and a fraction thereof is detected by a surface ionization detector to detect a tricyclic ring in the biological sample. A method for measuring a tricyclic antidepressant, which comprises detecting and quantifying an antidepressant. 2. The assay method according to claim 1, wherein the tricyclic antidepressant is a cibenzazepine derivative.